Thiolated nucleotide analogues for nucleic acid synthesis

ABSTRACT

The present disclosure provide systems, compositions, methods, reagents, kits and products for extending a nucleic acid that includes incorporating a nucleotide residue at a terminus of a nucleic acid using a polymerase enzyme and at least one nucleotide, wherein the at least one nucleotide includes a thiophosphate moiety, and wherein the at least one nucleotide is resistant to hydrolysis by phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of a phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of at least on chelation moiety that is configured to bind an orthophosphate moiety.

This application is a continuation application of U.S. application Ser.No.14/937,211, filed Nov. 10, 2015. U.S. application Ser. No.14/937,211claims benefit of U.S. Provisional Application No. 62/078,323, filed onNov. 11, 2014. All applications referenced in this section areincorporated by reference; each in its entirety.

BACKGROUND

The synthesis of nucleic acid polymers involves the enzyme-mediatedincorporation of individual nucleotides to form and extend a nucleicacid polymer. Nucleotides that are typically used in this process arenucleoside polyphosphates, such as deoxyribonucleotide triphosphates(dNTPs) which can be incorporated in the synthesis of deoxyribonucleicacids (DNA), and ribonucleotide triphosphates (NTPs) which can beincorporated in the synthesis of ribonucleic acids (RNA). Whenincorporated, nucleotides undergo hydrolysis of one or more of itsphosphodiester bonds, thus providing the thermodynamic driving force forthe overall reaction. For example, extension of a DNA strand with asingle dNTP can result in the DNA extended by the single nucleotide andthe release of pyrophosphate (PPi).

SUMMARY

This application relates to sulfur-containing (i.e., thiolated)analogues of nucleotides that are useful for nucleic acid synthesis, andmethods for using thiolated analogues of nucleotides.

In some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products for extending anucleic acid that includes incorporating a nucleotide residue at aterminus of a nucleic acid using a polymerase enzyme and at least onenucleotide, wherein the at least one nucleotide includes a thiophosphatemoiety, and wherein the at least one nucleotide is resistant tohydrolysis by phosphatase. In some embodiments, the incorporation of thenucleotide residue is template-directed. In some embodiments, thethiolated nucleotide is resistant to pyrophosphatase.

In some embodiments, the thiolated nucleotide is a deoxyribonucleotide.In some embodiments, the thiolated nucleotide is a deoxyribonucleotidethio-triphosphate. In some embodiments, the thiolated nucleotide is adeoxyribonucleotide-5′-γ[gamma]-thio-triphosphate.

In some embodiments, the incorporation of the nucleotide residue isperformed in the presence of a phosphatase. In some embodiments, thepolyphosphate leaving group produced upon incorporation of thenucleotide residue in the extended nucleic acid is hydrolyzed. In someembodiments, the pyrophosphate leaving group is a thio-pyrophosphate. Insome embodiments, the polyphosphate leaving group is athio-triphosphate. In some embodiments, the polyphosphate leaving groupis a thio-tetraphosphate.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products in which the specificrate of incorporation of the nucleotide with the thiophosphate moiety isat least 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97.5%, or at least 99% of the specificrate of incorporation of the analogous nucleotide without thethiophosphate moiety. In some embodiments, the rate of incorporation isbased on the rate of incorporation of the polymerase enzyme which is aBst polymerase.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products in which theincorporation of the nucleotide residue is performed in the presence ofat least one chelation moiety, wherein the chelation moiety isconfigured to bind an orthophosphate moiety. In some embodiments, thechelation moiety is configured to bind an orthophosphate moiety, whereinthe orthophosphate moiety is a monobasic orthophosphate, a dibasicorthophosphate, a tribasic orthophosphate, a monobasic thiophosphate, adibasic thiophosphate, or a tribasic thiophosphate.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products for identifying abase at a position in a target nucleic acid, including incorporating anucleotide residue at a terminus of an extension primer that ishybridized to the target nucleic acid using a polymerase enzyme and atleast one nucleotide, and identifying the position in a target nucleicacid based on the incorporation of the nucleotide residue, wherein theat least one nucleotide includes a thiophosphate moiety, and wherein thenucleotide is resistant to hydrolysis by phosphatase, wherein thenucleotide residue is incorporated when the nucleotide includes a basethat is complementary to the corresponding position in the targetnucleic acid.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products that include acomposition that includes a polymerase enzyme, at least one nucleotide,wherein the nucleotide includes a thiophosphate moiety and is resistantto hydrolysis by phosphatase. In some embodiments, the compositionincludes a phosphatase, such as pyrophosphatase or alkaline phosphatase.In some embodiments, the composition includes a nucleic acid and anextension primer complementary to at least a portion of the nucleicacid. In some embodiments, the composition includes at least onechelation moiety, wherein the chelation moiety is configured to bind anorthophosphate moiety.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products that include acompound that includes a nucleotide that includes a thiophosphate moietyand which is resistant to hydrolysis by phosphatase. In someembodiments, the nucleotide is resistant to hydrolysis bypyrophosphatase. In some embodiments, the nucleotide is adeoxyribonucleotide. In some embodiments, the nucleotide is adeoxyribonucleotide thio-triphosphate. In some embodiments, thenucleotide is a deoxyribonucleotide-5′-γ[gamma]-thio-triphosphate. Insome embodiments, the polymerase-mediated specific rate of incorporationof the nucleotide with the thiophosphate moiety is at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97.5%, or at least 99% of the polymerase-mediatedspecific rate of incorporation of the analogous nucleotide without thethiophosphate moiety. In some embodiments, the specific rate ofincorporation of the nucleotide with the thiophosphate moiety is thesame or greater than the specific rate of incorporation of the analogousnucleotide without the thiophosphate moiety. In some embodiments, therate of incorporation is based on the polymerase enzyme which is a Bstpolymerase.

The present disclosure provides methods, systems, compositions,reagents, kits and products for identifying a base at a position in atarget nucleic acid, comprising: (a) incorporating a nucleotide at aterminus of an extension primer that is hybridized to the target nucleicacid using a polymerase enzyme and at least one nucleotide, wherein theterminal phosphate of the at least one nucleotide includes athio-phosphate moiety, wherein the at least one nucleotide is resistantto hydrolysis by a phosphatase enzyme, and wherein the nucleotideincorporation produces a thio-pyrophosphate and a hydrogen ion or aproton; and (b) identifying the nucleotide that is incorporated at theterminus of the extension primer. In some embodiments, the at least onenucleotide is resistant to hydrolysis by a pyrophosphatase enzyme. Insome embodiments, the nucleotide incorporating step is performed in thepresence of a phosphatase enzyme and/or in the presence of apyrophosphatase enzyme. In some embodiments, the at least one nucleotidecomprises a deoxyribonucleotide-5′-γ[gamma]-thio-triphosphate. In someembodiments, the methods, systems, compositions, reagents, kits andproducts further comprises hydrolyzing the thio-pyrophosphate in thepresence of the phosphatase, thereby producing an orthophosphate. Insome embodiments, the methods, systems, compositions, reagents, kits andproducts further comprises identifying the nucleotide that isincorporated at the terminus of the extension primer by detecting thehydrogen ion or the proton. In some embodiments, the nucleotideincorporation step is conducted in the presence of at least onechelation moiety, wherein the chelation moiety is configured to bind theorthophosphate moiety. In some embodiments, the methods, systems,compositions, reagents, kits and products further comprises binding theat least one chelation moiety to the orthophosphate. In someembodiments, the orthophosphate is selected from a monobasicorthophosphate, a dibasic orthophosphate, a tribasic orthophosphate, amonobasic thiophosphate, a dibasic thiophosphate, and a tribasicthiophosphate. In some embodiments, the specific rate of incorporationof the nucleotide with the thiophosphate moiety is at least 95% of thespecific rate of incorporation of the analogous nucleotide without thethiophosphate moiety. In some embodiments, the polymerase enzyme is awild-type or mutant Bst polymerase enzyme. In some embodiments, theincorporating the nucleotide is conducted in a reaction chamber that isoperatively coupled at least one ion sensor that detects hydrogen ionsor protons. In some embodiments, the at least one ion sensor comprisesand ISFET. In some embodiments, the incorporating the nucleotide in step(a) is conducted on an array of reaction chambers, wherein individualreaction chambers in the array are operatively coupled to at least oneion sensor that detects hydrogen ions or protons. In some embodiments,the individual reaction chambers in the array are operatively coupled toat least one ISFET.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts schematically an exemplary nucleotide extension reactioninvolving the incorporation of a nucleotide in the presence of an enzymehaving nucleic acid polymerase activity.

FIG. 2 depicts schematically an exemplary nucleotide extension reactioninvolving the incorporation of a nucleotide in the presence of an enzymehaving nucleic acid polymerase activity and an enzyme having phosphataseactivity.

FIG. 3 depicts schematically an exemplary nucleotide extension reactioninvolving the incorporation of a nucleotide with a thiophosphate moietyin the presence of an enzyme having nucleic acid polymerase activity andan enzyme having phosphatase activity.

FIG. 4 depicts an exemplary phosphatase resistance assay of nucleotidetriphosphate and nucleotide gamma-S-triphosphate.

FIG. 5 depicts schematically an exemplary phosphate complexing agent.

DETAILED DESCRIPTION

The extension of nucleic acid polymers by the incorporation ofindividual nucleotides is a key feature of many processes in molecularbiology, both in natural and artificial contexts. Examples of the latterwhich entail steps of nucleic acid extension include nucleic acidamplification, quantitative polymerase chain reaction, and nucleic acidsequencing. Nucleic acid extension involves the incorporation of anucleotide residue by a polymerase into the nucleic acid that is beingextended. This incorporation and extension is typically mediated by anenzyme, such as a nucleic acid polymerase, and uses an activated versionof the nucleotide to be incorporated. An activated nucleotide typicallyincludes one or more high-energy chemical bonds, at least one of whichis broken and/or reformed as a lower energy bond, thus providing thefree energy to drive the extension reaction. In certain embodiments, theactivated chemical bond include, but are not limited to, phosphodiesterbonds, phosphoramidite bonds, phosphothioester bonds, phosphoraminebonds, and the like.

A particular application of nucleic acid extension reactions is in thefield of nucleic acid sequencing. In nearly all sequencing platforms,whether it be chemical degradation, chain-termination,sequence-by-synthesis, pyrosequencing, massively parallel,ion-sensitive, or single molecule platforms, the key step in determiningthe sequence of a target nucleic acid involves the extension of nucleicacid by incorporation of one or more nucleotides.

As shown in FIG. 1, an exemplary polymerase-mediated nucleic acidextension reaction is shown. A nucleotide triphosphate in the presenceof magnesium ion, polymerase enzyme, and the terminal end of a nucleicacid is incorporated at the terminus of the nucleic acid. Theincorporation reaction typically results in a release of a pyrophosphatemoiety and a hydrogen ion or proton. This reaction is a net exergonicreaction as a result of the free energy contribution by the hydrolysisof a phosphate diester bond.

As depicted in the schematic of the extension reaction, certainby-products are produced as a result of this extension reaction,specifically an equivalent each of hydrogen ion and pyrophosphate. Incertain embodiments, one or more of the products of the reaction can beused to detect or measure the extension reaction. For example, asdescribed herein, ion-based nucleic acid sequencing methods andplatforms include the Ion Torrent PGM™ or Proton™ sequencer (IonTorrent™ Systems, Life Technologies Corporation) the released hydrogenion can result in a pH change in the reaction environment, which can bedetected by an ion sensor. In certain embodiments, the extensionreaction is performed in a microwell, to which the ion sensor iscoupled. Advantages of performing the extension reaction in a definedspace such as a microwell include the ability to control the influx andefflux of reagents and by-products. Another advantage is the ability toperform a multiplicity of extension reaction equivalents in the samedefined space, which can produce a corresponding increase in equivalentsof reaction products. Particularly if the multiple extension reactionsare extensions of a homogenous population of template nucleic acids,such as would be performed in a sequencing reaction, the multipleparallel reactions will additively produce a larger signal to bedetected and measured.

One manner in which sequencing technology continuously advances isminiaturization. For example, by decreasing the physical footprint inwhich the nucleic acid extension reactions occurs, such as by decreasingthe size and/or volume of the microwell containing the extensionreaction, one can increase the number of reactions that can be performedwithin a given space or area on a platform or substrate. However, onedisadvantage that can arise from reducing the size in this manner isthat the quantity of extension reactions (e.g. the absolute number ofextension reactions) in each microwell can be reduced. In embodiments inwhich detection or measurement of the extension reaction is based ondetection or measurement of one or more by-products of the reaction,then this reduction can result in a decreased total signal to measurethe progress of the extension reactions.

A further effect of this reduced signal can be a reduction in theeffective read-length of each sequencing reaction. In embodiments inwhich each reaction involves sequential extension reaction, theefficiency and yield of each extension can decrease with each nucleotidethat is incorporated. As a result of the decreased efficiency, inconjunction with the decreased signal due to the reduced amount of thereaction, the effective read-length measured in each well can also bereduced.

Thus, one strategy to counter this decrease in total efficiencyresulting from a decreased number of reactions is to increase theefficiency of the extension reactions. In some embodiments, such as theextension reaction shown in FIG. 1, the schematically-depicted extensionreaction results in hydrogen ion and pyrophosphate as by-products.However, this extension reaction can be driven further exergonically byhydrolysis of a second high-energy diester bond in the pyrophosphateby-product, thereby increasing the net favorable free energy, and hencethe efficiency, of each nucleotide incorporation. An exemplaryembodiment of this reaction is shown in FIG. 2. As shown in thisexemplary schematic reaction, the nucleotide incorporation reactionshown in FIG. 1 is performed in the presence of an additional enzymehaving phosphatase and/or pyrophosphatase activity, which results in thehydrolysis of a second phosphodiester bond. This second hydrolysisincreases the net free energy of the reaction and can result in anincreased yield and efficiency of the first nucleotide incorporationstep.

Although the exemplary reaction depicted in FIG. 2 can result in anextension reaction having a higher efficiency of the overall nucleotideincorporation reaction, in certain contexts and embodiments thisapproach can have certain disadvantages. For example, sequencing methodsthat measure or detect the hydrogen ion by-product can be hindered bythe additional hydrolysis step as the orthophosphates produced asby-products in this reaction can act as conjugate bases, thusneutralizing any net production of hydrogen ions.

The schematic exemplary reaction of FIG. 2 also has other potentialdisadvantages in certain embodiments and contexts. For example, althoughthe hydrolysis by the phosphatase and/or pyrophosphatase activity canincrease the efficiency of the reaction as described above, thenucleotide polyphosphate starting reagent, such as the commonly usednucleotide triphosphates (dNTPs or NTPs), can also be susceptible tohydrolysis by the same phosphatase and/or pyrophosphatase activity.Thus, without isolating the nucleotide polyphosphates from thesubsequent phosphatase and/or pyrophosphatase activity, or modifying thenucleotide polyphosphate to render it resistant to said activity, thereaction as shown may not be able to be performed in a single reactionenvironment with all reagents and enzymes present simultaneously.Moreover, attempts to mitigate this problem by modification of thenucleotide polyphosphate to render it resistant to phosphatase and/orpyrophosphatase activity, such as by the addition of a protecting group,can also result in a nucleotide that may be a poorer substrate for thepolymerase enzyme.

In certain embodiments, the present disclosure relates to compounds,compositions, methods, systems, apparatus and kits that include anucleotide that includes at least one thiophosphate moiety. In certainembodiments, a thiophosphate moiety includes a molecular substructurehaving the formula of PS_(4-x)O_(x) ³⁻. In certain embodiments, athiophosphate moiety includes a molecular substructure having theformula of PSO₃ ³⁻, PS₂O₂ ³⁻, PS₃O³⁻, PS₃O³⁻, or phosphate-basedderivatives of any of the foregoing, including phosphoesters,phosphodiesters, phosphotriesters, phosphoamides, or phosphodiamides. Insome embodiments, the thiophosphate can include or be substituted withany one or more of the following, or any suitable combination thereof:one or more alkyl groups, one or more cycloalkyl groups, one or morearyl groups, one or more heteroaryl groups, one or more halogen groups,one or more amino groups, one or more alkylamino groups, one or moredialkylamino groups, one or more mercapto or thio groups, one or morealkylthio groups, or one or more cyclic derivatives of the foregoing(e.g., cycloalkyl groups), or any suitable combination thereof.

In certain embodiments, the thiolated nucleotide having a thiophosphatemoiety is a nucleoside polyphosphate, such as a nucleotide diphosphate,a nucleoside triphosphate, a nucleoside tetraphosphate, a nucleosidepentaphosphate, or nucleotides having 6,7, 8, 9 10 or more phosphates,or higher-order polyphosphates, in which one or more of the phosphatemoieties is a thiophosphate moiety. In certain embodiments, the sulfurof the thiophosphate moiety is connected only to the phosphate andoptionally a hydrogen. In certain embodiments, the sulfur of thethiophosphate moiety bridges two phosphate centers.

In certain embodiments, the sulfur is bonded to the phosphate mostproximate to the nucleoside, thus forming part of the α(alpha)-thiophosphate of the nucleoside polyphosphate. In certainembodiments, the sulfur is bonded to the phosphate that is second-mostproximate to the nucleoside, thus forming part of the β(beta)-thiophosphate of the nucleoside polyphosphate. In certainembodiments, the sulfur is bonded to the phosphate that is third-mostproximate to the nucleoside, thus forming part of the γ(gamma)-thiophosphate of the nucleoside polyphosphate. In certainembodiments, the sulfur is bonded to the phosphate that is fourth-mostproximate to the nucleoside, thus forming part of the δ(delta)-thiophosphate of the nucleoside polyphosphate. In certainembodiments, the sulfur is bonded to the phosphate that is fifth-mostproximate to the nucleoside, thus forming part of the ε(epsilon)-thiophosphate of the nucleoside. polyphosphate.

In certain embodiments, the sulfur is bonded to the phosphate that isthe terminal phosphate of the nucleoside polyphosphate.

In certain embodiments, the sulfur is bonded to the phosphate that isthird-most proximate to the nucleoside, thus forming part of the γ(gamma)-thiophosphate of the nucleoside polyphosphate, and which is alsothe terminal phosphate of the nucleoside thiophosphate. An example ofthis species is depicted in FIG. 3 in the form of a nucleoside γ(gamma)-thiophosphate

Without intending to be bound by theory, the thiolated nucleotides ofthe present disclosure are resistant to hydrolysis by enzymes havingphosphatase and/or pyrophosphatase activity. In certain embodiments,thiolated nucleotides in which the sulfur is bonded to the terminalpolyphosphate of a nucleoside polyphosphate are resistant to phosphataseor pyrophosphatase activity.

As an example of this resistant, representative results from anexemplary experiment are depicted in FIG. 4. This figure shows thenormalized fluorescence intensity over time of two different nucleosidepolyphosphates, γ-S-thymidinyl-triphosphate (“gamma STTP) andthymidinyl-triphosphate (“TTP”) in the presence or absence of calfintestinal alkaline phosphatase (“CIP”). Phosphatase-mediated hydrolysisis reflected as a non-increasing level of fluorescence, whereasphosphatase resistance is reflected as an increase in normalizedfluorescence intensity. Therefore, as depicted in this exemplaryexperiment, the thiolated nucleotide showed a resistance to phosphatasethat was not reflected in the analogous non-thiolated nucleotide, TTP.

In certain embodiments, the thiolated nucleotides of the presentdisclosure are incorporated in nucleic acid extension reactions bypolymerase enzymes at the same or substantially the same rate and/oraffinity. In some embodiments, the polymerase-mediated specific rate ofincorporation of the nucleotide with the thiophosphate moiety is atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97.5%, or at least 99% of thepolymerase-mediated specific rate of incorporation of the analogousnucleotide without the thiophosphate moiety. In some embodiments, thespecific rate of incorporation of the nucleotide with the thiophosphatemoiety is the same or greater than the specific rate of incorporation ofthe analogous nucleotide without the thiophosphate moiety. In someembodiments, the rate of incorporation is based on the polymerase enzymewhich is a Bst polymerase. Without intending to be bound by theory,thiolated nucleotides of the present disclosure are incorporated at thesame or substantially the same rate as their non-thiolated counterpartbecause the substitution of an oxygen atom for a sulfur atom isrelatively non-disruptive with respect to steric and polarityproperties. Moreover, the thiolated nucleotides in which the sulfur ispresent at the terminal (e.g., gamma) position of the polyphosphatewould mean that this substitution is the most distal from thealpha-phosphate where the incorporation reaction is concerned.

In some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products for extending anucleic acid that includes incorporating a nucleotide residue at aterminus of a nucleic acid using a polymerase enzyme and at least onenucleotide, wherein the at least one nucleotide includes a thiophosphatemoiety, and wherein the at least one nucleotide is resistant tohydrolysis by phosphatase. In some embodiments, the incorporation of thenucleotide residue is template-directed. In some embodiments, thethiolated nucleotide is resistant to pyrophosphatase.

In some embodiments, the thiolated nucleotide is a deoxyribonucleotide.In some embodiments, the thiolated nucleotide is a deoxyribonucleotidethio-triphosphate. In some embodiments, the thiolated nucleotide is adeoxyribonucleotide-5′-γ[gamma]-thio-triphosphate.

In some embodiments, the incorporation of the nucleotide residue isperformed in the presence of a phosphatase. In some embodiments, thepolyphosphate leaving group produced upon incorporation of thenucleotide residue in the extended nucleic acid is hydrolyzed. In someembodiments, the pyrophosphate leaving group is a thio-pyrophosphate.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products in which the specificrate of incorporation of the nucleotide with the thiophosphate moiety isat least 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97.5%, or at least 99% of the specificrate of incorporation of the analogous nucleotide without thethiophosphate moiety. In some embodiments, the rate of incorporation isbased on the rate of incorporation of the polymerase enzyme which is aBst polymerase.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products in which theincorporation of the nucleotide residue is performed in the presence ofat least one chelation moiety, wherein the chelation moiety isconfigured to bind an orthophosphate moiety. In some embodiments, thechelation moiety is configured to bind preferentially to anorthophosphate moiety compared to a pyrophosphate or thiopyrophosphatemoiety. In some embodiments, the chelation moiety is configured to bindan orthophosphate moiety, wherein the orthophosphate moiety is amonobasic orthophosphate, a dibasic orthophosphate, a tribasicorthophosphate, a monobasic thiophosphate, a dibasic thiophosphate, or atribasic thiophosphate.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products for identifying abase at a position in a target nucleic acid, including incorporating anucleotide residue at a terminus of an extension primer that ishybridized to the target nucleic acid using a polymerase enzyme and atleast one nucleotide, and identifying the position in a target nucleicacid based on the incorporation of the nucleotide residue, wherein theat least one nucleotide includes a thiophosphate moiety, and wherein thenucleotide is resistant to hydrolysis by phosphatase, wherein thenucleotide residue is incorporated when the nucleotide includes a basethat is complementary to the corresponding position in the targetnucleic acid.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products that include acomposition that includes a polymerase enzyme, at least one nucleotide,wherein the nucleotide includes a thiophosphate moiety and is resistantto hydrolysis by phosphatase. In some embodiments, the compositionincludes a phosphatase, such as pyrophosphatase. In some embodiments,the composition includes a nucleic acid and an extension primercomplementary to at least a portion of the nucleic acid. In someembodiments, the composition includes at least one chelation moiety,wherein the chelation moiety is configured to bind an orthophosphatemoiety.

In the some embodiments, the present teachings provide systems,compositions, methods, reagents, kits and products that include acompound that includes a nucleotide that includes a thiophosphate moietyand which is resistant to hydrolysis by phosphatase. In someembodiments, the nucleotide is resistant to hydrolysis bypyrophosphatase. In some embodiments, the nucleotide is adeoxyribonucleotide. In some embodiments, the nucleotide is adeoxyribonucleotide thio-triphosphate. In some embodiments, thenucleotide is a deoxyribonucleotide-5′-γ[gamma]-thio-triphosphate. Insome embodiments, the polymerase-mediated specific rate of incorporationof the nucleotide with the thiophosphate moiety is at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97.5%, or at least 99% of the polymerase-mediatedspecific rate of incorporation of the analogous nucleotide without thethiophosphate moiety. In some embodiments, the specific rate ofincorporation of the nucleotide with the thiophosphate moiety is thesame or greater than the specific rate of incorporation of the analogousnucleotide without the thiophosphate moiety. In some embodiments, therate of incorporation is based on the polymerase enzyme which is a Bstpolymerase

In certain embodiments, the present description relates to otherimprovements to enhance the efficiency of the nucleotide incorporationreactions used in, for example, nucleic acid extension reactions used innucleic acid sequencing analysis as described herein. In certainembodiments, such improvements can be used independently or inconjunction with other improved described herein or known in the art.

As discussed herein, the efficiency of a nucleotide incorporationreaction, such as when used as part of a nucleic acid extensionreaction, can be enhanced by removing by-products of the reaction, suchas by hydrolysis of the pyrophosphate or thiopyrophosphate by-products.In this manner, the net favorable energetics of the overall reaction areincreased by the additional hydrolysis step. Another analogous approachis to sequester or otherwise remove one or more by-products of thereaction. Without intending to be bound by theory, sequestration ofby-products may improve the overall reaction efficiency by shifting theequilibrium of the reaction towards completion by decreasing theconcentration of the sequestered by-products. Moreover, sequestration ofone or more by-products can also prevent or limit the participation ofthe by-product in side-reactions that would otherwise diminish thedetection or measurement of other by products. For example, as describedherein, certain sequencing technologies rely on detection or measurementof the hydrogen ions generated during the nucleic acid extensionreaction. However, as described herein, the generation of orthophosphateor thiophosphate moieties, particularly those having basic equivalents,may absorb the hydrogen ions produced during the reaction. Withoutintending to be bound by theory, by sequestering orthophosphate orthiophosphate moieties, particularly when such moieties are in theirbasic forms, can limit or prevent their neutralization of the hydrogenions by their basic phosphate or thiophosphate equivalents.

As an example of these embodiments, the present disclosure includes theuse of one or more complexing moieties that is configured to bind aphosphate moiety, a thiophosphate moiety, or analogues thereof. Forexample, as depicted in FIG. 5, an exemplary complexing molecule that iscapable of binding a phosphate or thiophosphate moiety.

In certain embodiments, complexing agents of the present disclosure havepreferred affinities for monobasic, dibasic, or tribasic orthophosphate.In certain preferred embodiments, complexing agents have preferredaffinities for tribasic orthophosphate (PO₄ ³⁻), which may release thehighest number of equivalents of hydrogen ions.

In certain embodiments, complexing agents of the present disclosure havepreferred affinities for monobasic, dibasic, or tribasic thiophosphate.In certain preferred embodiments, complexing agents have preferredaffinities for tribasic orthophosphate (PSO₃ ³⁻), which may release thehighest number of equivalents of hydrogen ions.

In certain embodiments, complexing agents of the present disclosure areable to bind their preferred orthophosphate or thiophosphate species ata pH at which the nucleotide extension reaction is performed. In thismanner, the sequestration of the phosphate or thiophosphate species canoccur in substantially real-time with the generation of the phosphate orthiophosphate from the reaction.

In certain embodiments, complexing agents of the present disclosure havea low affinity for the nucleoside polyphosphate or nucleoside thiolatedpolyphosphate, or a lower affinity for the nucleoside polyphosphate ornucleoside thiolated polyphosphate as compared to orthophosphate orthiophosphate.

A nucleotide comprises any compound that can bind selectively to, or canbe polymerized by, a polymerase. Typically, but not necessarily,selective binding of the nucleotide to the polymerase is followed bypolymerization of the nucleotide into a nucleic acid strand by thepolymerase; occasionally however the nucleotide may dissociate from thepolymerase without becoming incorporated into the nucleic acid strand,an event referred to herein as a “non-productive” event. A nucleotidepolymerization reaction (also called a “nucleotide incorporation”reaction) can include primer extension reactions, nucleic acidamplification reactions, or sequence-by-synthesis reactions. Nucleotidesinclude not only naturally occurring nucleotides but also any analogs,regardless of their structure, that can bind selectively to, or can bepolymerized by, a polymerase. While naturally occurring nucleotidestypically comprise base, sugar and phosphate moieties, the nucleotidesof the present disclosure can include compounds lacking any one, some orall of such moieties. In some embodiments, the nucleotide can optionallyinclude a chain of phosphorus atoms comprising three, four, five, six,seven, eight, nine, ten or more phosphorus atoms. In some embodiments,the phosphorus chain can be attached to any carbon of a sugar ring, suchas the 5′ carbon. The phosphorus chain can be linked to the sugar withan intervening O or S. In some embodiments, one or more phosphorus atomsin the chain can be part of a phosphate group having P and O. In someembodiments, the phosphorus atoms in the chain can be linked togetherwith intervening O, NH, S, methylene, substituted methylene, ethylene,substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where Rcan be a 4-pyridine or 1-imidazole). In some embodiments, the phosphorusatoms in the chain can have at least one side group including O, BH₃, orS. In the phosphorus chain, a phosphorus atom with a side group otherthan O can be a substituted phosphate group. In the phosphorus chain,phosphorus atoms with an intervening atom other than O can be asubstituted phosphate group. Some examples of nucleotide analogs aredescribed in

Some examples of nucleotides that can be used in the disclosed methodsand compositions include, but are not limited to, ribonucleotides,deoxyribonucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, ribonucleotide polyphosphates, deoxyribonucleotidepolyphosphates, modified ribonucleotide polyphosphates, modifieddeoxyribonucleotide polyphosphates, peptide nucleotides, modifiedpeptide nucleotides, metallonucleosides, phosphonate nucleosides, andmodified phosphate-sugar backbone nucleotides, analogs, derivatives, orvariants of the foregoing compounds. In some embodiments, the nucleotidecan comprise non-oxygen moieties such as, for example, thio- orborano-moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof.

In some embodiments, a nucleotide can include a purine or pyrimidinebase, including adenine, guanine, cytosine, thymine or uracil. In someembodiments, a nucleotide includes dATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the nucleotide is unlabeled. In some embodiments,the nucleotide comprises a label and referred to herein as a “labelednucleotide”. In some embodiments, the label can be attached to anyportion of a nucleotide including a base, sugar or any interveningphosphate group or a terminal phosphate group, i.e., the phosphate groupmost distal from the sugar.

In some embodiments, a nucleotide (or analog thereof) can be attached toa label. In some embodiments, a label comprises a detectable moiety. Insome embodiments, a label can generate, or cause to generate, adetectable signal. A detectable signal can be generated from a chemicalor physical change (e.g., heat, light, electrical, pH, saltconcentration, enzymatic activity, or proximity events). For example, aproximity event can include two reporter moieties approaching eachother, or associating with each other, or binding each other. Adetectable signal can be detected optically, electrically, chemically,enzymatically, thermally, or via mass spectroscopy or Ramanspectroscopy. A label can include compounds that are luminescent,photoluminescent, electroluminescent, bioluminescent, chemiluminescent,fluorescent, phosphorescent or electrochemical. A label can includecompounds that are fluorophores, chromophores, radioisotopes, haptens,affinity tags, atoms or enzymes. In some embodiments, the labelcomprises a moiety not typically present in naturally occurringnucleotides. For example, the label can include fluorescent, luminescentor radioactive moieties.

By way of a non-limiting example of nucleotide incorporation (e.g., DNApolymerization), the steps or events of nucleotide incorporation arewell known and generally comprise: (1) complementary base-pairing atemplate DNA molecule with a DNA primer molecule having a terminal 3′ OH(the terminal 3′ OH provides the polymerization initiation site for DNApolymerase); (2) binding the base-paired template/primer duplex with aDNA-dependent polymerase to form a complex; (3) a candidate nucleotidebinds with the DNA polymerase which interrogates the candidatenucleotide for complementarity with the template nucleotide on thetemplate DNA molecule; (4) the DNA polymerase may undergo aconformational change (e.g., from an open to a closed complex if thecandidate nucleotide is complementary); (5) the polymerase catalyzesnucleotide incorporation.

In one embodiment, the polymerase catalyzes nucleotide incorporation byforming a bond between the candidate nucleotide and the nucleotide atthe terminal end of the polymerization initiation site. The polymerasecan catalyze the terminal 3′ OH of the primer exerting a nucleophilicattack on the bond between the α and β phosphates of the candidatenucleotide to mediate a nucleotidyl transferase reaction resulting inphosphodiester bond formation between the terminal 3′ end of the primerand the candidate nucleotide (i.e., nucleotide incorporation in atemplate-dependent manner), and concomitant cleavage to form a cleavageproduct. The polymerase can liberate the cleavage product. In someembodiments, where the polymerase incorporates a nucleotide havingphosphate groups, the cleavage product includes one or more phosphategroups. In some embodiments, where the polymerase incorporates anucleotide having substituted phosphate groups, the cleavage product mayinclude one or more substituted phosphate groups. In some embodiments,nucleotide incorporation reactions produce one or more cleavage products(e.g., byproducts) including polyphosphate compounds (pyrophosphates),hydrogen ions, or protons.

The candidate nucleotide may or may not be complementary to the templatenucleotide on the template molecule. The candidate nucleotide can bindthe polymerase and then dissociate from the polymerase. If thenucleotide dissociates from the polymerase (e.g., it is notincorporated), it can be liberated and typically carries intactpolyphosphate groups.

In some embodiments, nucleotide incorporation can be a reversetranscriptase reaction which includes a nucleic acid template (RNA orDNA), primers, nucleotides (or analogs thereof) and reversetranscriptase enzyme. In some embodiments, nucleotide incorporation canbe a transcription reaction which includes an RNA template, nucleotides(or analogs thereof) and a DNA-dependent RNA polymerase enzyme.Nucleotide incorporation events involving reverse transcriptase orDNA-dependent RNA polymerase are well known in the art.

In some embodiments, a nucleotide incorporation reaction can includenatural nucleotides, nucleotide analogs, or a combination of both.

A polymerase comprises any enzyme that can catalyze the polymerizationof nucleotides (including analogs thereof) into a nucleic acid strand.Typically but not necessarily such nucleotide polymerization can occurin a template-dependent fashion. Such polymerases can include withoutlimitation naturally occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases, chemicallymodified polymerases, synthetic molecules or assemblies, and anyanalogs, derivatives or fragments thereof that retain the ability tocatalyze such polymerization. Optionally, the polymerase can be a mutantpolymerase comprising one or more mutations involving the replacement ofone or more amino acids with other amino acids, the insertion ordeletion of one or more amino acids from the polymerase, or the linkageof parts of two or more polymerases. The term “polymerase” and itsvariants, as used herein, also refers to fusion proteins comprising atleast two portions linked to each other, where the first portioncomprises a peptide that can catalyze the polymerization of nucleotidesinto a nucleic acid strand and is linked to a second portion thatcomprises a second polypeptide, such as, for example, a reporter enzymeor a processivity-enhancing domain. In some embodiments, a polymerasecan be a high fidelity polymerase. Typically, the polymerase comprisesone or more active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. In some embodiments, a polymeraseincludes or lacks other enzymatic activities, such as for example, 3′ to5′ exonuclease activity, 5′ to 3′ exonuclease activity, or stranddisplacement activity. In some embodiments, a polymerase can be isolatedfrom a cell, or generated using recombinant DNA technology or chemicalsynthesis methods. In some embodiments, a polymerase can be expressed inprokaryote, eukaryote, viral, or phage organisms. In some embodiments, apolymerase can be post-translationally modified proteins or fragmentsthereof.

In some embodiments, the polymerase can include any one or morepolymerases, or biologically active fragment of a polymerase, which aredescribed in any of: U.S. published application No. 2011/0262903,published Oct. 27, 2011; International PCT Publication No. WO2013/023176, published Feb. 14, 2013; International PCT Publication No.WO 2013/023176, published Feb. 14, 2013; U.S. 61/884,921, filed Sep. 30,2013; U.S. published application No. 2011/0262903, published Oct. 27,2011; U.S. published application No. 2011/0301041, published Dec. 8,2011; U.S. published application No. 2012/0202276; U.S. Ser. No.13/035177, filed Feb. 25, 2011, and published as U.S. publishedapplication No. 2011/0318748 on Dec. 29, 2011; U.S. Ser. No. 13/572,488,filed Aug. 10, 2012; and U.S. 61/884,921, filed Sep. 30, 2013.

In some embodiments, a polymerase can be a DNA polymerase and includewithout limitation bacterial DNA polymerases, eukaryotic DNApolymerases, archaeal DNA polymerases, viral DNA polymerases and phageDNA polymerases.

In some embodiments, a polymerase can be a replicase, DNA-dependentpolymerase, primases, RNA-dependent polymerase (including RNA-dependentDNA polymerases such as, for example, reverse transcriptases), athermo-labile polymerase, or a thermo-stable polymerase. In someembodiments, a polymerase can be any Family A or B type polymerase. Manytypes of Family A (e.g., E. coli Pol I), B (e.g., E. coli Pol II), C(e.g., E. coli Pol III), D (e.g., Euryarchaeotic Pol II), X (e.g., humanPol beta), and Y (e.g., E. coli UmuC/DinB and eukaryotic RAD30/xerodermapigmentosum variants) polymerases are described in Rothwell and Watsman2005 Advances in Protein Chemistry 71:401-440. In some embodiments, apolymerase can be a T3, T5, T7, or SP6 RNA polymerase.

In some embodiments, nucleotide incorporation reactions can be conductedwith one type or a mixture of different types of polymerases. In someembodiments, nucleotide incorporation reactions can be conducted with alow fidelity or high fidelity polymerase.

In some embodiments, an archaeal DNA polymerase can be, withoutlimitation, an A family DNA polymerase; a B family DNA polymerase; amixed-type polymerase; an unclassified DNA polymerase and RT familypolymerase; and variants and derivatives thereof.

In some embodiments, nucleic acid amplification reactions can becatalyzed by heat-stable or heat-labile polymerases.

In some embodiments, an archaeal DNA polymerase can be, withoutlimitation, a thermostable or thermophilic DNA polymerase such as, forexample: a Bacillus subtilis (Bsu) DNA polymerase I large fragment; aThermus aquaticus (Taq) DNA polymerase; a Thermus filiformis (Tfi) DNApolymerase; a Phi29 DNA polymerase; a Bacillus stearothermophilus (Bst)DNA polymerase; a Thermococcus sp. 9° N-7 DNA polymerase; a Bacillussmithii (Bsm) DNA polymerase large fragment; a Thermococcus litoralis(Tli) DNA polymerase or Vent™ (exo-) DNA polymerase (from New EnglandBiolabs); or “Deep Vent” (exo-) DNA polymerase (New England Biolabs).

In some embodiments, the DNA polymerase is an A family DNA polymeraseselected from the group consisting of a Pol I-type DNA polymerase suchas E. coli DNA polymerase, the Klenow fragment of E. coli DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Platinum Taq DNApolymerase series, T7 DNA polymerase, and Tth DNA polymerase. In someembodiments, the DNA polymerase is Bst DNA polymerase. In otherembodiments, the DNA polymerase is E. coli DNA polymerase. In someembodiments, the DNA polymerase is the Klenow fragment of E. coli DNApolymerase. In some embodiments, the polymerase is Taq DNA polymerase.In some embodiments, the polymerase is T7 DNA polymerase.

In other embodiments, the DNA polymerase is a B family DNA polymeraseselected from the group consisting of Tli polymerase, Pfu polymerase,Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase,Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENTpolymerase, phage Phi29 polymerase, and phage B103 polymerase. In someembodiments, the polymerase is phage Phi29 DNA polymerase. In someembodiments the polymerase is phage B103 polymerase, including, forexample, the variants disclosed in U.S. Patent Publication No.2011/0014612.

In other embodiments, the DNA polymerase is a mixed-type polymeraseselected from the group consisting of EX-Taq polymerase, LA-Taqpolymerase, Expand polymerase series, and Hi-Fi polymerase. In yet otherembodiments, the DNA polymerase is an unclassified DNA polymeraseselected from the group consisting of Tbr polymerase, Tfl polymerase,Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, and Tfi polymerase.

In other embodiments, the DNA polymerase is an RT polymerase selectedfrom the group consisting of HIV reverse transcriptase, M-MLV reversetranscriptase and AMV reverse transcriptase. In some embodiments, thepolymerase is HIV reverse transcriptase or a fragment thereof having DNApolymerase activity.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses forincorporating one or more nucleotides (e.g., nucleotide analogs),comprising: providing a template nucleic acid hybridized to a primer andbound to a polymerase; synthesizing a new nucleic acid strand byincorporating one or more nucleosides (e.g., any of the nucleotideanalogs described in the present teachings) sequentially at the 3′ endof the primer; and detecting such incorporation at the 3′ end of theprimer. The incorporation nucleotide can be detected by measuring aconcentration of a hydrogen ion byproduct generated if the incorporatednucleoside is complementary to the corresponding nucleotides in thetemplate nucleic acid.

In some embodiments, the polymerase can include any one or more of theamino acid substitutions described herein. In some embodiments, at leastone of the one or more amino acid substitutions can be a conservativeamino acid substitution. In some embodiments, each of the one or moreamino acid substitutions is a conservative amino acid substitution.

In some embodiments, the polymerase includes any one of the modifiedpolymerases described herein. In some embodiments, the polymerase is abufferless polymerase. For example, the polymerase can have reducedbuffering capacity relative to the corresponding unsubstitutedpolymerase.

In some embodiments, the polymerase includes one or more amino acidsubstitutions that substantially remove the buffering capacity of thepolymerase within the pH range of about 4 to about 10 relative to thecorresponding unsubstituted polymerase. The unsubstituted polymerase canbe the wild-type version of the polymerase.

In some embodiments, the one or more amino acid substitutions in thepolymerase substantially remove the buffering capacity of the polymeraserelative to the corresponding unsubstituted polymerase within the rangeof about pH 7 to about pH 9. The unsubstituted polymerase can be thewild-type version of the polymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the polymerase is a conservative amino acidsubstitution that is selected from the group consisting of histidine toarginine, glutamic acid to glutamine, aspartic acid to asparagine,lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 4.0 to about 10.0 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7 to about 9 with another amino acidresidue. In some embodiments, the pKa of the amino acid residue is asolution pKa of the amino acid residue. In other embodiments, the pKa ofthe amino acid residue is a pKa of the amino acid residue in the contextof the corresponding wild-type protein.

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 4.0 and about 10.0 with an amino acid residuehaving a pKa that is greater than about 10.0 or less than about 4.0. Infurther embodiments the amino acid residue having a pKa that is greaterthan about 10.0 or less than about 4.0 is selected from the groupconsisting of: Arg, Asp, Gln, Lys, Ile, Leu, Norleucine (Nle), Met, Phe,Ser, Thr, Trp, Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more conservative aminosubstitutions includes a substitution of an amino acid residue having apKa of between about 7 and about 9 with an amino acid residue having apKa that is greater than about 9 or less than about 7. In furtherembodiments the amino acid residue having a pKa that is greater thanabout 9 or less than about 7 is selected from the group consisting of:Arg, Asp, Gln, ly, Ile, Leu, Norleucine (Nle), Met, Phe, Ser, Thr, Trp,Val and N-terminal Formylmethionine (N-fMet).

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 6.0 to about 8.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 6.0 and about 8.0 with an amino acid residue havinga pKa that is greater than about 8.0 or less than about 6.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa within the range of about 7.0 to about 9.0 with another amino acidresidue.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue having apKa of between about 7.0 and about 9.0 with an amino acid residue havinga pKa that is greater than about 9.0 or less than about 7.0.

In some embodiments, at least one of the one or more amino acidsubstitutions includes a substitution of an amino acid residue selectedfrom the group consisting of His, Glu, Asp, Tyr, and Lys with anotheramino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that reduce the buffering capacity of theprotein relative to the corresponding wild-type protein within the rangeof about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, or about pH7 to about pH 9. In some embodiments, at least one of the one or moreamino acid substitutions includes a substitution of an amino acidresidue that is at least 20%, at least 25%, at least 30%, at least 35%or at least 40% solvent exposed in the corresponding wild-type proteinwith another amino acid residue.

In some embodiments, at least one of the one or more amino acidsubstitutions is a substitution of an amino acid residue with an alanineresidue.

In some embodiments, the polymerase comprises one or more amino acidconservative amino acid substitutions that reduce the buffering capacityof the protein relative to the corresponding wild-type protein withinthe range of about pH 4 to about pH 10, about pH 5.5 to about pH 9.5, orabout pH 7 to about pH 9. In some embodiments, at least one of the oneor more amino acid substitutions includes a substitution of an aminoacid residue that is at least 20%, at least 25%, at least 30%, at least35% or at least 40% solvent exposed in the corresponding wild-typeprotein with another amino acid residue.

In some embodiments, the polymerase comprises one or more conservativeamino acid substitutions that substantially remove the bufferingcapacity of the polymerase within the range of about pH 4 to about pH10, about pH 5.5 to about pH 9.5, or about pH 7 to about pH 9.

In some embodiments, the DNA polymerase is a Bst DNA polymerasecomprising one or more amino acid substitutions that substantiallyreduce its buffering capacity within the range of about pH 4 to about pH10. In some embodiments, the one or more amino acid substitutionssubstantially remove the buffering capacity of the polymerase within therange of about pH 4 to about pH 10. For example, see Table 3.

In some embodiments, the one or more amino acid substitutions in the BstDNA polymerase substantially reduce the buffering capacity of thepolymerase relative to the corresponding unsubstituted polymerase withinthe range of about pH 7 to about pH 9. In some embodiments, the one ormore amino acid substitutions substantially remove the bufferingcapacity of the Bst polymerase within the range of about pH 7 to aboutpH 9. In some embodiments, the one or more amino acid substitutionssubstantially reduce the buffering capacity of the Bst polymeraserelative to the corresponding unsubstituted Bst polymerase within therange of about pH 7 to about pH 9. In some embodiments, theunsubstituted polymerase can be the wild-type version of the Bstpolymerase.

In some embodiments, at least one of the one or more amino acidsubstitutions in the Bst DNA polymerase is a conservative amino acidsubstitution. In further embodiments, the at least one conservativeamino acid substitution is selected from the group consisting ofhistidine to arginine, glutamic acid to glutamine, aspartic acid toasparagine, lysine to arginine, and tyrosine to phenylalanine.

In some embodiments, the one or more conservative amino acidsubstitutions are of one or more amino acid residues shown in Table 2.In some embodiments, the one or more conservative amino acidsubstitutions are selected from the group consisting of H46R, H273R,H281R, E446Q, H473R, H528R, H572R and Y477F, the numbering of amino acidresidues being in accordance with that of SEQ ID NO: 1.

In some embodiments, the one or more amino acid substitutions includes asubstitution of alanine at position 2 with Met, Asn, Gln, Leu, Ile, Phe,or Trp, the numbering of amino acid residues being in accordance withthat of SEQ ID NO:2.

In some embodiments, the Bst DNA polymerase comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID NO: 4, or a variant thereof having one or more conservativeamino acid substitutions. In some embodiments, the Bst DNA polymerasecomprises the amino acid sequence of SEQ ID NO: 2. In some embodiments,the Bst DNA polymerase is a variant of a protein comprising the aminoacid sequence shown in SEQ ID NO: 2, wherein the variant comprises anamino acid sequence that is at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 2.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 3. In some embodiments, the Bst DNA polymerase isa variant of a protein comprising the amino acid sequence shown in SEQID NO: 3, wherein the variant comprises an amino acid sequence that isat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 3.

In some embodiments, the Bst DNA polymerase comprises the amino acidsequence of SEQ ID NO: 4. In other embodiments, the Bst polymerase is avariant of a protein comprising the amino acid sequence shown in SEQ IDNO: 4, wherein the variant comprises an amino acid sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 4.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a Therminator™ DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially remove its buffering capacity within the range of about pH7 to about pH 9, wherein the one or more conservative amino acidsubstitutions are selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table5.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table6.

In some embodiments, the DNA polymerase is a KOD DNA polymerasecomprising one or more amino acid substitutions that substantiallyremove its buffering capacity within the range of about pH 7 to about pH9. The one or more conservative amino acid substitutions are optionallyselected from the group consisting of: histidine to arginine, glutamicacid to glutamine, lysine to arginine and tyrosine to phenylalanine. Insome embodiments, the one or more conservative amino acid substitutionsare of one or more amino acid residues shown in Table 6.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 7 toabout pH 9. The unsubstituted polymerase can be the wild-type version ofthe polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In some embodiments, the DNA polymerase is a B103 DNA polymerasecomprising one or more conservative amino acid substitutions thatsubstantially reduce its buffering capacity relative to thecorresponding unsubstituted polymerase within the range of about pH 4 toabout pH 10. The unsubstituted polymerase can be the wild-type versionof the polymerase. The one or more conservative amino acid substitutionsare optionally selected from the group consisting of: histidine toarginine, glutamic acid to glutamine, lysine to arginine and tyrosine tophenylalanine. In some embodiments, the one or more conservative aminoacid substitutions are of one or more amino acid residues shown in Table7.

In other embodiments of the method, the DNA polymerase is a B103 DNApolymerase comprising one or more conservative amino acid substitutionsthat substantially remove its buffering capacity within the range ofabout pH 7 to about pH 9. The one or more conservative amino acidsubstitutions are optionally selected from the group consisting of:histidine to arginine, glutamic acid to glutamine, lysine to arginineand tyrosine to phenylalanine. In some embodiments, the one or moreconservative amino acid substitutions are of one or more amino acidresidues shown in Table 7.

In some embodiments, the disclosure relates generally to a method forperforming a nucleotide polymerization reaction comprising contacting amodified polymerase or a biologically active fragment thereof with anucleic acid template in the presence of one or more nucleotides (e.g.,any of the nucleotide analogues described herein), where the modifiedpolymerase or the biologically active fragment thereof includes one ormore amino acid modifications relative to a reference polymerase andwhere the modified polymerase or the biologically active fragmentthereof has an increased dissociation time constant relative to thereference polymerase, and polymerizing at least one of the one or morenucleotides using the modified polymerase or the biologically activefragment thereof. In some embodiments, the method includes polymerizingat least one of the one or more nucleotides using the modifiedpolymerase or the biologically active fragment thereof in the presenceof a high ionic strength solution. In some embodiments, the method canfurther include polymerizing the at least one nucleotide in atemplate-dependent fashion. In some embodiments, the modified polymeraseor the biologically active fragment thereof comprises or consists of atleast 80% identity to SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQID NO: 36, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 45.In some embodiments, the modified polymerase or the biologically activefragment thereof comprises at least 25 contiguous amino acids from thepolymerase catalytic domain. In some embodiments, the modifiedpolymerase or the biologically active fragment thereof comprises atleast 25 contiguous amino acids from the polymerase DNA binding domain.In some embodiments, the modified polymerase or the biologically activefragment thereof comprises or consists of at least 100 amino acidresidues having at least 80% identity to SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 45. In someembodiments, the modified polymerase or the biologically active fragmentthereof comprises or consists of at least 150 amino acid residues of thepolymerase catalytic domain having at least 90% identity to SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ IDNO: 45.

In some embodiments, the disclosure generally relates to a polymerase ora biologically active fragment thereof having DNA polymerase activityand at least 80% identity to SEQ ID NO: 22, SEQ ID NO: 36, SEQ ID NO:37, or SEQ ID NO: 39.

In some embodiments, disclosure is generally related to an isolated andpurified polypeptide comprising or consisting of a recombinantpolymerase homologous to SEQ ID NO: 22 or biologically active fragmentthereof comprises any one or more mutations relative to SEQ ID NO: 22selected from the group consisting of: N31R, N31K, D77K, D77H, D113N,D114R, D130A, D130H, D144M, D144K, L212A, E220K, N234R, N234K, V241K,V251K, D264Q, D264S, D264K, Y272R, H273R, L280R, H281A, H281M, E294S,E294F, E294G, E294K, V299K, V299H, V299F, D303R, I331Q, E325R, L335T,E336P, I354W, I354F, I370A, Q409R, G416K, V418M, V418I, G420K, D423S,D423K, D423N, D423R, D423T, D423G, D423I, D423K, G425R, Q428W, N429R,N429K, E446Q, F448K, N457T, A462T, H473R, Y477F, D480R, D480F, D480H,D480A, D480S, D480N, D480Q, N485W, N485Y, N487H, N487W, N487F, N487I,V488R, E493Q, M495Q, H528A, H528R, H528K,V533I, H572R, W577Y and D579F.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 22 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 22 or a biologically active fragment thereof and where therecombinant polymerase comprises a mutation or combination of mutationsrelative to SEQ ID NO: 22 selected from H46R, and where the polymerasefurther includes a mutation at one or more of E446Q, H572R, H273R,H281A, H473R, Y477F, D480R, or H528A.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 22 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 22 or a biologically active fragment thereof and where therecombinant polymerase comprises a mutation or combination of mutationsrelative to SEQ ID NO: 22 selected from E446Q, where the polymerasefurther includes a mutation at one or more of H46R, H572R, H273R, H281A,H473R, Y477F, D480R, or H528A.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 22 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 22 or a biologically active fragment thereof and where therecombinant polymerase comprises a mutation or combination of mutationsrelative to SEQ ID NO: 22 selected from H572R, where the polymerasefurther includes a mutation at one or more of E446Q, H572R, H273R,H281A, H473R, Y477F, D480R, or H528A.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 22 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 22 or a biologically active fragment thereof and where therecombinant polymerase comprises a C93 mutation.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 22 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 22 or a biologically active fragment thereof and where therecombinant polymerase comprises a Q238 mutation.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 22 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 22 or a biologically active fragment thereof and where therecombinant polymerase comprises a H273 mutation.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 22 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 22 or a biologically active fragment thereof and where therecombinant polymerase comprises a H281 mutation.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 22 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 22 or a biologically active fragment thereof and where therecombinant polymerase comprises a H473 mutation.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 22 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 22 or a biologically active fragment thereof and where therecombinant polymerase comprises a H528 mutation.

In some embodiments, disclosure is generally related to an isolated andpurified polypeptide comprising or consisting of at least 80% identityto SEQ ID NO: 23 and having one or more amino acid mutations selectedfrom the group consisting of N31R, N31K, D77K, D77H, D113N, D114R,D130A, D130H, D144M, D144K, L212A, E220K, N234R, N234K, V241K, V251K,D264Q, D264S, D264K, Y272R, H273R, L280R, H281A, E294S, E294F, E294G,E294K, V299K, V299H, V299F, D303R, I331Q, E325R, L335T, E336P, I354W,I354F, I370A, Q409R, G416K, V418M, V418I, G420K, D423S, D423K, D423N,D423R, D423T, D423G, D423I, D423K, G425R, Q428W, N429R, N429K, F448K,N457T, A462T, H473R, Y477F, D480R, D480F, D480H, D480A, D480S, D480N,D480Q, N485W, N485Y, N487H, N487W, N487F, N487I, V488R, E493Q, M495Q,H528A, V533I, W577Y and D579F.

In some embodiments, disclosure is generally related to an isolated andpurified polypeptide comprising or consisting of at least 80% identityto SEQ ID NO: 36 and having one or more amino acid mutations selectedfrom the group consisting of E471K, N485R, R492K, D513K, A675K, D732R,S739W, V740R and E745Q.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 36 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 36 or a biologically active fragment thereof and where therecombinant polymerase comprises a mutation or combination of mutationsrelative to SEQ ID NO: 36 selected from E471K , wherein the polymerasefurther includes a mutation at one or more of: N485R, R492K, D513K,A675K, D732R, S739W, V740R and E745Q.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 36 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 36 or a biologically active fragment thereof and where therecombinant polymerase comprises a mutation or combination of mutationsrelative to SEQ ID NO: 36 selected from V740R, wherein the polymerasefurther includes a mutation at one or more of: E471K, N485R, D513K andE745Q.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 36 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 36 or a biologically active fragment thereof and where therecombinant polymerase comprises a N485 mutation.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 36 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 36 or a biologically active fragment thereof and where therecombinant polymerase comprises a mutation or combination of mutationsrelative to SEQ ID NO: 36 selected from a D513 mutation.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 36 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 36 or a biologically active fragment thereof and where therecombinant polymerase comprises a mutation or combination of mutationsrelative to SEQ ID NO: 36 selected from a D732 mutation.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 36 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 36 or a biologically active fragment thereof and where therecombinant polymerase comprises a mutation or combination of mutationsrelative to SEQ ID NO: 36 selected from an E745 mutation.

In some embodiments, the reference polymerase has or comprises the aminoacid sequence of SEQ ID NO: 36, and the modified polymerase has orcomprises the amino acid sequence of the reference polymerase. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to the amino acid sequenceof the reference polymerase. In some embodiments, the modifiedpolymerase further includes any one or more amino acid mutationsselected from the group consisting of: N326R, N326K, D372K, D372H,D408N, D409R, D425A, D425H, D439M, D439K, L507A, E515K, N529R, N529K,V536K, V546K, D559Q, D559S, Y567R, L575R, E589S, E589F, E589G, V594K,V594H, V594F, D598R, I626Q, L630T, E631P, I649W, I649F, I665A, Q704R,G711K, V713M, V713I, G715K, D718S, D718K, D718N, D718R, D718T, D718G,D718I, D718K, G720R, Q723W, N724R, N724K, F743K, N752T, A757T, D775R,D775F, D775H, D775A, D775S, D775N, D775Q, N780W, N780Y, N782H, N782W,N782F, N782I, E782Q, V783R, E788Q, M790Q, V828I, W872Y and D874F,wherein the numbering is relative of the amino acid sequence of SEQ IDNO: 37.

In some embodiments, the reference polymerase has or comprises the aminoacid sequence of SEQ ID NO: 37, and the modified polymerase has orcomprises the amino acid sequence of the reference polymerase. In someembodiments, the modified polymerase comprises an amino acid sequencethat is at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to the amino acid sequenceof the reference polymerase. In some embodiments, the modifiedpolymerase further includes any one or more amino acid mutationsselected from the group consisting of: N326R, N326K, H341R, D372K,D372H, C388R, D408N, D409R, D425A, D425H, D439M, D439K, L507A, E515K,N529R, N529K, V536K, V546K, Q533C, D559Q, D559S, Y567R, H568R, L575R,H576A, E589S, E589F, E589G, V594K, V594H, V594F, D598R, I626Q, L630T,E631P, I649W, I649F, I665A, Q704R, G711K, V713M, V713I, G715K, D718S,D718K, D718N, D718R, D718T, D718G, D718I, D718K, G720R, Q723W, N724R,N724K, E741Q, F743K, N752T, A757T, H768R, Y772F, D775R, D775F, D775H,D775A, D775S, D775N, D775Q, N780W, N780Y, N782H, N782W, N782F, N782I,E782Q, V783R, E788Q, M790Q, H823A,V828I, C845Q, H867R, W872Y and D874F,wherein the numbering is relative of the amino acid sequence of SEQ IDNO: 37.

In some embodiments, disclosure is generally related to an isolated andpurified polypeptide comprising or consisting of at least 80% identityto SEQ ID NO: 39 and having one or more amino acid mutations selectedfrom the group consisting of E245K, S259R, T266K, E290K, A448K, D505R,A512W, R513R and E518Q.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:39 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 39 or a biologically fragment thereof and wherethe recombinant polymerase comprises a mutation or combination ofmutations relative to SEQ ID NO: 39 selected from E245K, where thepolymerase further includes a mutation at one or more of: S259R, T266K,E290K, A448K, D505R, A512W, R513R and E518Q.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:39 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 39 or a biologically fragment thereof and wherethe recombinant polymerase comprises a mutation or combination ofmutations relative to SEQ ID NO: 39 selected from D505R, where thepolymerase further includes a mutation at one or more of: E245K, S259R,T266K, E290K, A448K, A512W, R513R and E518Q.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:39 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 39 or a biologically fragment thereof and wherethe recombinant polymerase comprises an E290 mutation.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:39 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 39 or a biologically fragment thereof and wherethe recombinant polymerase comprises an 5259 mutation.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:39 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 39 or a biologically fragment thereof and wherethe recombinant polymerase comprises an R513 mutation.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:39 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 39 or a biologically fragment thereof and wherethe recombinant polymerase comprises an A512 mutation.

In some embodiments, the disclosure relates generally to a method forperforming a nucleotide polymerization reaction comprising or consistingof contacting a modified polymerase or a biologically active fragmentthereof with a nucleic acid template in the presence of one or morenucleotides (e.g., any of the nucleotide analogues described herein),where the modified polymerase or the biologically active fragmentthereof includes one or more amino acid modifications relative to areference polymerase and where the modified polymerase or thebiologically active fragment thereof has a lowered systematic error,decreased strand bias, increased raw read accuracy and/or increasedtotal sequencing throughput as compared to the reference polymerase, andpolymerizing at least one of the one or more nucleotides using themodified polymerase or the biologically active fragment thereof.

In some embodiments, the isolated or modified polymerases as disclosedherein can include a fusion of a first naturally occurring polymerasedomain (e.g., a catalytic domain) with a first genetically engineeredpolymerase domain (e.g., a binding domain). In some embodiments, theisolated or modified polymerases disclosed herein can include a fusionof a first genetically engineered polymerase domain (e.g., a catalyticdomain) to a second genetically engineered polymerase domain (e.g., abinding domain), thereby forming an isolated or modified polymeraseretaining polymerase activity.

In some embodiments, the modified polymerase or the biologically activefragment thereof comprises or consists of at least 80% identity, or atleast 90% identity, or at least 95% identity, or at least 98% identity,or at least 99% identity to SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ IDNO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQID NO: 81 or SEQ ID NO: 82.

In some embodiments, the modified polymerase or the biologically activefragment thereof comprises at least 25 contiguous amino acids from thepolymerase catalytic domain. In some embodiments, the modifiedpolymerase or the biologically active fragment thereof comprises atleast 25 contiguous amino acids from the polymerase DNA binding domain.In some embodiments, the modified polymerase or the biologically activefragment thereof comprises or consists of at least 100 amino acidresidues having at least 80% identity to SEQ ID NO: 47, SEQ ID NO: 48,SEQ ID NO: 49, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ IDNO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, themodified polymerase or the biologically active fragment thereofcomprises or consists of at least 150 amino acid residues of thepolymerase catalytic domain having at least 90% identity to SEQ ID NO:47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 64, SEQ ID NO: 65, SEQ IDNO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82.

In some embodiments, the method includes amplifying conditions having ahigh ionic strength solution. In some embodiments, amplifying conditionshaving a high ionic strength solution include at least 120 mM salt. Insome embodiments, amplifying conditions having a high ionic strengthsolution include 125 mM salt. In some embodiments, amplifying conditionshaving a high ionic strength solution include 125 mM to 200 mM salt. Insome embodiments, the salt can include KCl and/or NaCl.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising or consisting of at least 80%identity to SEQ ID NO: 46 or a biologically active fragment thereof andhaving one or more amino acid mutations (e.g., substitutions) selectedfrom the group consisting of N31R, N31K, H46R, D77K, D77H, D113N, D114R,D130A, D130H, D144M, D144K, L212A, E220K, N234R, N234K, V241K, V251K,A263K, D264A, D264R, D264Q, D264S, D264K, Y272R, H273N, H273R, L280R,H281A, H281M, E294S, E294F, E294G, E294K, V299K, V299H, V299F, D303R,I331Q, E325R, L335T, E336P, I354W, I354F, I370A, Q409R, G416K, V418M,V418I, G420K, D423S, D423K, D423N, D423R, D423T, D423G, D423I, D423K,G425R, Q428W, N429R, N429K, E446Q, F448K, N457T, A462T, H473R, Y477F,D480R, D480F, D480H, D480A, D480S, D480N, D480Q, N485K N485W, N485Y,N487H, N487R N487W, N487F, N4871, V488R, E493Q, E493R, M495Q, H528A,H528F, H528S, V533I, H572R, W577Y and D579F, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 46.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising or consisting of at least 80%identity to SEQ ID NO: 47 or a biologically active fragment thereof andhaving one or more amino acid mutations (e.g., substitutions) selectedfrom the group consisting of N31R, N31K, D77K, D77H, D113N, D114R,D130A, D130H, D144M, D144K, L212A, E220K, N234R, N234K, V241K, V251K,D264A, D264R, D264Q, D264S, D264K, Y272R, H273N, H273R, L280R, H281A,H281M, E294S, E294F, E294G, E294K, V299K, V299H, V299F, D303R, I331Q,E325R, L335T, E336P, I354W, I354F, I370A, Q409R, G416K, V418M, V418I,G420K, D423S, D423K, D423N, D423R, D423T, D423G, D423I, D423K, G425R,Q428W, N429R, N429K, F448K, N457T, A462T, H473R, Y477F, D480R, D480F,D480H, D480A, D480S, D480N, D480Q, N485K N485W, N485Y, N487H, N487RN487W, N487F, N4871, V488R, E493Q, E493R, M495Q, H528A, H528F, H528S,V533I, W577Y and D579F, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 47.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide having at least 90% identity to SEQ ID NO: 47and includes an amino acid substitution at one or more positionscorresponding to positions selected from the group consisting of: N487,N485, E493, A263, D264, H528, H273, D423, D480, H281, E220 and N234,wherein the numbering is relative to SEQ ID NO: 47.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising or consisting of at least 80%identity to SEQ ID NO: 60 or a biologically active fragment thereof andhaving one or more amino acid mutations selected from the groupconsisting of E471K, N485R, R492K, D513K, A675K, D732R, S739W, V740R andE745Q, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 60.

In some embodiments, the recombinant polymerase homologous to SEQ ID NO:60, comprises a mutation or combination of mutations relative to SEQ IDNO: 60 selected from any one or more of: E471K, N485R, R492K, D513K,A675K, D732R, S739W, V740R and E745Q.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising or consisting of at least 80%identity to SEQ ID NO: 61 or a biologically active fragment thereof andhaving one or more amino acid mutations (e.g., substitutions) selectedfrom any one or more of: N782R, N780K, E788R, A558K, D559A, D559R,H823S, H823F, H568N, D718K, D775R, H576M, E515K and N529R, wherein thenumbering is relative to the amino acid sequence of SEQ ID NO: 61.

In some embodiments, the recombinant polymerase homologous to SEQ ID NO:61, comprises a mutation or combination of mutations relative to SEQ IDNO: 61 selected from any one or more of: H341R, C388R, Q533C, H568R,H576A, E741Q, H768R, Y772F, H823A, C845Q,H867R, N782R, N780K, E788R,A558K, D559A, D559R, H823S, H823F, H568N, D718K, D775R, H576M, E515K andN529R, wherein the numbering is relative to the amino acid sequence ofSEQ ID NO: 61.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising or consisting of at least 80%identity to SEQ ID NO: 63 or a biologically active fragment thereof andhaving one or more amino acid mutations selected from the groupconsisting of E245K, S259R, T266K, E290K, A448K, D505R, A512W, R513R andE518Q, wherein the numbering is relative to SEQ ID NO: 63.

In some embodiments, the recombinant polymerase homologous to SEQ ID NO:63, comprises a mutation or combination of mutations relative to SEQ IDNO: 63 selected from any one of more of: E245K, S259R, T266K, E290K,A448K, D505R, A512W, and E518Q, wherein the numbering is relative to SEQID NO: 63.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising or consisting of at least 90%identity to SEQ ID NO: 70 and includes an amino acid substitution at oneor more positions corresponding to positions selected from the groupconsisting of: N780K, E788R, A558K, D559A, D559R, H823S, H823F, H568N,D718K, D775R, H576M, E515K and N529R, wherein the numbering is relativeto SEQ ID NO: 70.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising or consisting of at least 80%identity to SEQ ID NO: 71 or a biologically active fragment thereof andhaving one or more amino acid mutations selected from the groupconsisting of N780K, E788R, A558K, D559A, D559R, H823S, H823F, D775R,H576M, H568N, E515K and N529R, wherein the numbering is relative to theamino acid sequence of SEQ ID NO: 71.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising or consisting of at least 80%identity to SEQ ID NO: 72 or a biologically active fragment thereof andhaving one or more amino acid mutations selected from the groupconsisting of N780K, E788R, A558K, D559A, D559R, H823S, H823F, D775R,H576M, E515K and N529R, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 72.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising at least 90% identity to SEQ ID NO:73 and includes an amino acid substitution at one or more positionscorresponding to positions selected from the group consisting of: N780K,E788R, A558K, D559A, D559R, H823S, H823F, D718K, D775R, H568N, E515K andN529R, wherein the numbering is relative to SEQ ID NO: 73.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising or consisting of at least 80%identity to SEQ ID NO: 74 or a biologically active fragment thereof andhaving one or more amino acid mutations selected from the groupconsisting of N780K, E788R, A558K, D559A, D559R, D718K, D775R, H576M,H568N, E515K and N529R, wherein the numbering is relative to the aminoacid sequence of SEQ ID NO: 74.

In some embodiments, the disclosure is generally related to an isolatedand purified polypeptide comprising at least 90% identity to SEQ ID NO:82 and includes an amino acid substitution at one or more positionscorresponding to positions selected from the group consisting of: N485,E493, A263, D264, H528, H273, D423 and D480, wherein the numbering isrelative to SEQ ID NO: 82.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 46 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 46 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 46 comprises a mutationor combination of mutations relative to SEQ ID NO: 46 selected fromH46R, and where the polymerase further includes a mutation at one ormore of E446Q, H572R, H273R, H281A, H473R, Y477F, D480R, or H528A,wherein the numbering is relative to SEQ ID NO: 46.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 46 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 46 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 46 comprises a mutationor combination of mutations relative to SEQ ID NO: 1 selected fromE446Q, where the polymerase further includes a mutation at one or moreof H46R, H572R, H273R, H281A, H473R, Y477F, D480R, or H528A, wherein thenumbering is relative to SEQ ID NO: 46.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 46 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 46 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 46 comprises a mutationor combination of mutations relative to SEQ ID NO: 46 selected fromH572R, where the polymerase further includes a mutation at one or moreof H46R, E446Q, H572R, H273R, H281A, H473R, Y477F, D480R, or H528A,wherein the numbering is relative to SEQ ID NO: 46.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 47 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 47 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 47 comprises a mutationor combination of mutations relative to SEQ ID NO: 47 selected fromN487R, and where the recombinant polymerase further includes a mutationat one or more of H281M, D423K, H273N, E493R, and D264A, wherein thenumbering is relative to SEQ ID NO: 47.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 47 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 47 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 47 comprises a mutationor combination of mutations relative to SEQ ID NO: 47 selected fromH281M, where the recombinant polymerase further includes a mutation atone or more of N487R, D264A, H273N and E493R, wherein the numbering isrelative to SEQ ID NO: 47.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 47 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 47 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 47 comprises a mutationor combination of mutations relative to SEQ ID NO: 47 selected fromE493R where the recombinant polymerase further includes a mutation atone or more of N487R, H281M, D423K, D264A, or H273N, wherein thenumbering is relative to SEQ ID NO: 47.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 60 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 60 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 60 comprises a mutationor combination of mutations relative to SEQ ID NO: 60 selected fromE471K , wherein the polymerase further includes a mutation at one ormore of: N485R, R492K, D513K, A675K, D732R, S739W, V740R and E745Q,wherein the numbering is relative to SEQ ID NO: 60.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 60 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 60 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 60 comprises a mutationor combination of mutations relative to SEQ ID NO: 60 selected fromV740R, wherein the polymerase further includes a mutation at one or moreof: E471K, N485R, D513K and E745Q, wherein the numbering is relative toSEQ ID NO: 60.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 61 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 61 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 61 comprises a mutationor combination of mutations relative to SEQ ID NO: 61 selected fromN782R, and where the recombinant polymerase further includes a mutationat one or more of N780K, E788R, A558K, D559A, D559R, H823S, H823F,H568N, D718K, D775R, E515K, N529R, or H576M, wherein the numbering isrelative to SEQ ID NO: 61.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 61 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 61 or a biologically active fragment thereof and where therecombinant polymerase comprises a mutation or combination of mutationsrelative to SEQ ID NO: 61 selected from N780K, where the recombinantpolymerase further includes a mutation at one or more of N782R, E788R,A558K, D559A, D559R, H823S, H823F, H568N, D718K, D775R, E515K, N529R, orH576M, wherein the numbering is relative to SEQ ID NO: 61.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 61 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 61 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 61 comprises a mutationor combination of mutations relative to SEQ ID NO: 61 selected fromE788R where the recombinant polymerase further includes a mutation atone or more of N782R, N780K, A558K, D559A, D559R, H823S, H823F, H568N,D718K, D775R, E515K, N529R, or H576M, wherein the numbering is relativeto SEQ ID NO: 61.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:63 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 63 or a biologically fragment thereof and wherethe recombinant polymerase homologous to SEQ ID NO: 63 comprises amutation or combination of mutations relative to SEQ ID NO: 63 selectedfrom E245K, where the polymerase further includes a mutation at one ormore of: S259R, T266K, E290K, A448K, D505R, A512W, R513R and E518Q,wherein the numbering is relative to SEQ ID NO: 63.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:63 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 63 or a biologically fragment thereof and wherethe recombinant polymerase homologous to SEQ ID NO: 63 comprises amutation or combination of mutations relative to SEQ ID NO: 63 selectedfrom E245K, where the polymerase further includes a mutation at one ormore of: S259R, T266K, E290K, A448K, D505R, A512W, R513R and E518Q,wherein the numbering is relative to SEQ ID NO: 63.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:63 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 63 or a biologically fragment thereof and wherethe recombinant polymerase homologous to SEQ ID NO: 63 comprises amutation or combination of mutations relative to SEQ ID NO: 63 selectedfrom D505R, where the polymerase further includes a mutation at one ormore of: E245K, S259R, T266K, E290K, A448K, A512W, R513R and E518Q,wherein the numbering is relative to SEQ ID NO: 63.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 70 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 70 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 70 comprises a mutationor combination of mutations relative to SEQ ID NO: 70 selected fromD718K, wherein the polymerase further includes a mutation at one or moreof: N780K, E788R, A558K, D559A, D559R, H823S, H823F, H568N, D775R, andH576M, wherein the numbering is relative to SEQ ID NO: 70.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 70 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 70 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 70 comprises a mutationor combination of mutations relative to SEQ ID NO: 70 selected fromH568N, wherein the polymerase further includes a mutation at one or moreof: N780K, E788R, A558K, D559A, D559R, H823S, H823F, D718K, D775R, orH576M, wherein the numbering is relative to SEQ ID NO: 70.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 71 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 71 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 71 comprises a mutationor combination of mutations relative to SEQ ID NO: 71 selected fromH568N, where the polymerase further includes a mutation at one or moreof N780K, E788R, A558K, D559A, D559R, H823S, H823F, D775R, or H576M,wherein the numbering is relative to SEQ ID NO: 71.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:73 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 73 or a biologically fragment thereof and wherethe recombinant polymerase homologous to SEQ ID NO: 73 comprises amutation or combination of mutations relative to SEQ ID NO: 73 selectedfrom D718K, where the polymerase further includes a mutation at one ormore of: A558K, H823S, H823F, D559A, D559R, D568N, D775R, E788R, N780K,E515K and N529R, wherein the numbering is relative to SEQ ID NO: 73.

In some embodiments, the disclosure is generally related to acomposition comprising a recombinant polymerase homologous to SEQ ID NO:74 or a biologically active fragment thereof having at least 80%identity to SEQ ID NO: 74 or a biologically fragment thereof and wherethe recombinant polymerase comprises a mutation or combination ofmutations relative to SEQ ID NO: 74 selected from D718K, where thepolymerase further includes a mutation at one or more of: A558K, D559A,D559R, D568N, D775R, E788R, H576M, N780K, E515K and N529R, wherein thenumbering is relative to SEQ ID NO: 74.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 75 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 75 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 75 comprises a mutationor combination of mutations relative to SEQ ID NO: 75 selected fromN782R, wherein the polymerase further includes a mutation at one or moreof A558K, D559A, D559R, H823S, H823F, H568N, and H576M, wherein thenumbering is relative to SEQ ID NO: 75.

In some embodiments, the disclosure generally relates to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 75 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 75 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 75 comprises a mutationor combination of mutations relative to SEQ ID NO: 75 selected fromH568N, wherein the polymerase further includes a mutation at one or moreof: N780K, E788R, A558K, D559A, D559R, H823S, H823F, D775R, or H576M,wherein the numbering is relative to SEQ ID NO: 75.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 76 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 76 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 76 comprises a mutationor combination of mutations relative to SEQ ID NO: 76 selected fromH568N, where the polymerase further includes a mutation at one or moreof N780K, E788R, A558K, D559A, D559R, H823S, H823F, D775R, or H576M,wherein the numbering is relative to SEQ ID NO: 76.

In some embodiments, the disclosure relates generally to a compositioncomprising a recombinant polymerase homologous to SEQ ID NO: 77 or abiologically active fragment thereof having at least 80% identity to SEQID NO: 77 or a biologically active fragment thereof and where therecombinant polymerase homologous to SEQ ID NO: 77 comprises a mutationor combination of mutations relative to SEQ ID NO: 77 selected fromD718K, where the polymerase further includes a mutation at one or moreof N780K, A558K, H823S, H823F, or D775R, wherein the numbering isrelative to SEQ ID NO: 77.

In some embodiments, the disclosure relates generally to an isolated andpurified polypeptide (e.g., a fusion polypeptide) comprising orconsisting of at least 90% identity to SEQ ID NO: 70, SEQ ID NO: 71, SEQID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76,SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO:81 or SEQ ID NO: 82, or a biologically active fragment thereof and canoptionally further include one or more amino acid mutations selectedfrom the group consisting of E515K and N529R, wherein the numbering isrelative to the amino acid sequence of SEQ ID NO: 61. In someembodiments, the recombinant polymerase homologous to SEQ ID NO: 61, SEQID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74,SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77 can optionally furtherinclude one or more mutations selected from the group consisting ofE515K and N529R, wherein the numbering is relative to SEQ ID NO: 61.

In some embodiments, a template nucleic acid is hybridized to a primerand bound to a polymerase, where the template nucleic acid comprises asingle-stranded or double-stranded polynucleotide, or a mixture of both.In some embodiments, the polynucleotide includes a plurality ofpolynucleotides having the same or different sequences. In someembodiments, the plurality of polynucleotides have the same or differentlengths. In some embodiments, the plurality of polynucleotides comprisesabout 2-10, or about 10-50, or about 50-100, or about 100-500, or about500-1,000, or about 1,000-5,000, or about 10³-10⁶, or about 10⁶-10¹⁰, ormore different polynucleotides. In some embodiments, a plurality ofpolynucleotides comprises polymers of deoxyribonucleotides,ribonucleotides, and/or analogs thereof. In some embodiments, aplurality of polynucleotides comprises naturally-occurring, synthetic,recombinant, cloned, amplified, unamplified or archived (e.g.,preserved) forms. In some embodiments, a plurality of polynucleotidescomprises DNA, cDNA RNA or chimeric RNA/DNA, and nucleic acid analogs.

In some embodiments, a template nucleic acid comprises any nucleic acid,including DNA, cDNA, RNA, or RNA/DNA hybrids. The template nucleic acidcan be single-stranded or double-stranded nucleic acids, and can haveany length. The template nucleic acid can be chromosomal, genomic,transcriptomic, organellar, methylated, chromatin-linked, cloned,unamplified, amplified, natural or synthetic, and can be isolated fromany source (for example, from an organism, normal or diseased cells ortissues, body fluids, archived tissue (e.g., tissue archived in formalinand/or in paraffin). In some embodiments, the template nucleic acid canbe isolated from any source including prokaryotes, eukaryotes (e.g.,humans, plants and animals), fungus, and viruses; cells; tissues; normalor diseased cells or tissues or organs, body fluids including blood,urine, serum, lymph, tumor, saliva, anal and vaginal secretions,amniotic samples, perspiration, and semen; environmental samples;culture samples; or synthesized nucleic acid molecules prepared usingrecombinant molecular biology or chemical synthesis methods. In someembodiments, the template nucleic acid can be isolated from aformalin-fixed tissue, or from a paraffin-embedded tissue, or from aformalin-fix paraffin-embedded (FFPE) tissue. In some embodiments, atemplate molecule can be about 100bp-1000 bp, or about 1 kb-50 kb, orabout 50 kb-100 kb, or longer.

In some embodiments, the template nucleic acid includes at least oneprimer binding site. The template nucleic acid can be generated byjoining together an initial polynucleotide (from any source) to anucleic acid adaptor having a primer binding sequence. For example, theinitial polynucleotide and adaptor can be joined by ligation,hybridization or primer extension methods. An adapter can be joined toat least one end of a linear template, or within the body of a linear orcircular initial polynucleotide. Optionally, the template can becircularized after the adapter is joined.

In some embodiments, primers comprise polymers of deoxyribonucleotides,ribonucleotides, and/or analogs thereof. In some embodiments, primerscomprise naturally-occurring, synthetic, recombinant, cloned, amplified,or unamplified forms. In some embodiments, primers comprise DNA, cDNARNA, chimeric RNA/DNA, or nucleic acid analogs. In some embodiments,primers comprise single-stranded or double-stranded forms.

In some embodiments, at least a portion of a primer can hybridize with aportion of at least one strand of a template polynucleotide in thereaction mixture. In some embodiments, at least a portion of a primercan be partially or fully complementary to a portion of the templatepolynucleotide. A template polynucleotide can include a polynucleotidesequence of interest, or a nucleic acid adaptor sequence joined to thepolynucleotide sequence of interest.

In some embodiments, a primer can include or lack a terminal 3′OH whichcan serve as an initiation site for nucleotide incorporation. In someembodiments, a primer can include a terminal 3′ blocking group that doesnot serve as an initiation site for nucleotide incorporation.

In some embodiments, primers can be any length, including about 5-20nucleotides, or about 20-40 nucleotides, or about 40-60 nucleotides, orabout 60-80 nucleotides, or longer.

In some embodiments, a primer can have a 5′ or 3′ overhang tail (tailedprimer) that does not hybridize with a portion of at least one strand ofa template polynucleotide. In some embodiments, a non-complementaryportion of a tailed primer can be any length, including 1-50 or morenucleotides in length.

In some embodiments, a plurality of primers includes individual primersthat are essentially the same or are different. For example, primers inthe plurality can have essentially the same sequences or differentsequences, or can have essentially the same length or different lengths,or can include natural or synthetic forms or a mixture of both.

In some embodiments, a reaction mixture can contain at least one reagentfor conducting a nucleotide incorporation reaction. For example, areaction mixture can include any one or any combination of reagents: atleast one nucleotide, one or more polymerases, at least one templatemolecule, at least one primer, at least one divalent cation. Optionally,a reaction mixture can include other enzymes, including at least onephosphatase. Optionally, a reaction mixture can include at least oneaccessory protein that can: bind single-stranded or double-strandednucleic acids; mediate loading other protein onto a nucleic acid; unwindnucleic acid substrates; relax nucleic acids; resolve nucleic acidstructures; disassemble complexes of nucleic acids and proteins, ordisassemble nucleic acid structures; or hydrolyze nucleic acids. In someembodiments, an accessory protein comprises a sliding clamp protein. Insome embodiments, an accessory protein comprises a multimeric proteincomplex. In some embodiments, a multimeric protein complex comprises 2,3, 4, 5, 6, 7, 8, or more subunits. In some embodiments, a multimericaccessory protein complex comprises a homo-meric or hetero-meric proteincomplex.

Optionally, a reaction mixture includes one or more additives forenhancing nucleotide incorporation, including betaine, DMSO, proline,trehalose, MMNO (4-methylmorpholine N-oxide) or a PEG-like compound.

In some embodiments, methods for nucleotide incorporation reactions canbe conducted under conditions that are suitable for: binding anucleotide to a polymerase (where the polymerase is bound to a duplexthat includes a template molecule and primer); incorporating thenucleotide into the primer; generating at least one cleavage productsfrom nucleotide incorporation; detecting one or more cleavage products,or one or any combination of these steps.

In some embodiments, suitable conditions include well known parameters,such as: time, temperature, pH, buffers, reagents, cations, salts,co-factors, nucleotides, nucleic acids, and enzymes. In someembodiments, a reagent or buffer can include a source of ions, such asKCl, K-acetate, NH₄-acetate, K-glutamate, NH₄Cl, or ammonium sulfate. Insome embodiments, a reagent or buffer can include a source of divalentions, such as Mg²⁺ or Mn²⁺, MgCl₂, MnCl₂, or Mg-acetate. In someembodiments, a reagent or buffer can include magnesium, manganese and/orcalcium. In some embodiments, a buffer can include Tris, Tricine, HEPES,MOPS, ACES, MES, or inorganic buffers such as phosphate or acetate-basedbuffers which can provide a pH range of about 4-12. In some embodiments,a buffer can include chelating agents such as EDTA or EGTA. In someembodiments, a buffer can include dithiothreitol (DTT), glycerol,spermidine, and/or BSA (bovine serum albumin). In some embodiments, abuffer can include ATP.

In some embodiments, suitable conditions include conducting a nucleotideincorporation reaction in a liquid phase, including an aqueous fluid orimmiscible fluid. In some embodiments, a nucleotide incorporationreaction can be conducted in a continuous aqueous phase, or in ahydrophilic phase of an emulsion having a discontinuous hydrophilicphase and a continuous hydrophobic phase. In some embodiments, anaqueous fluid can be water-based. In some embodiments, a hydrophobicphase can be oil-based. In some embodiments, different nucleotideincorporation reactions can be conducted in separate compartments (e.g.,droplets) forming part of a hydrophilic phase of an emulsion having adiscontinuous hydrophilic phase and a continuous hydrophobic phase.

In some embodiments, suitable conditions include conducting a nucleotideincorporation reaction with a polymerase enzyme and one or morenucleotides.

In some embodiments, suitable conditions include cyclical temperaturechanges, or essentially isothermal temperature conditions, or acombination of both. In some embodiments, a reaction can be conducted ata temperature range of about 0-10° C., or about 10-20° C., or about20-30° C., or about 30-40° C., or about 40-50° C., or about 50-60° C.,or about 60-70° C., or about 70-80° C., or about 80-90° C., or about90-100° C., or high temperatures.

In some embodiments, suitable conditions include conducting a reactionfor a time, such as about 10-30 seconds, or about 30-60 seconds, orabout 1-3 minutes, or about 3-5 minutes, or about 5-6 minutes, or about6-7 minutes, or about 7-8 minutes, or about 8-9 minutes, or about 9-10minutes, or about 10-11 minutes, or about 11-12 minutes, or about 12-13minutes, or about 13-14 minutes, or about 14-15 minutes, or about 15-20minutes, or about 20-30 minutes, or about 30-45 minutes, or about 45-60minutes, or about 1-3 hours, or about 3-6 hours, or about 6-10 hours, orlonger.

In some embodiments, suitable conditions include conducting a reactionin a volume of about 1-10 uL, or about 10-25 uL, or about 25-50 uL, orabout 50-75 uL, or about 75-100 uL, or about 100-125 uL, or about125-150 uL, or about 150-200 uL, or more.

In some embodiments, suitable conditions include conducting a reactionin a tube or well. In some embodiments, the well can be a part of a96-well plate.

In some embodiments, methods for nucleotide incorporation comprise oneor more surfaces. In some embodiments, a surface can be attached with aplurality of first primers, the first primers of the plurality sharing acommon first primer sequence. In some embodiments, a surface can beattached with a plurality of first and second primers, where the firstand second primers have different sequences.

In some embodiments, a surface can be an outer or top-most layer orboundary of an object. In some embodiments, a surface can be interior tothe boundary of an object.

In some embodiments, a surface can be porous, semi-porous or non-porous.In some embodiments, a surface can be a planar surface, as well asconcave, convex, or any combination thereof. In some embodiments, asurface can be a bead, particle, microparticle, sphere, filter,flowcell, well, groove, channel reservoir, gel or inner wall of acapillary. In some embodiments, a surface includes the inner walls of acapillary, a channel, a well, groove, channel, reservoir. In someembodiments, a surface can include texture (e.g., etched, cavitated,pores, three-dimensional scaffolds or bumps).

In some embodiments, particles can have a shape that is spherical,hemispherical, cylindrical, barrel-shaped, toroidal, rod-like,disc-like, conical, triangular, cubical, polygonal, tubular, wire-likeor irregular.

In some embodiments, a surface can be made from any material, includingglass, polymers, borosilicate glass, silica, quartz, fused quartz, mica,polyacrylamide, plastic polystyrene, polycarbonate, polymethacrylate(PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS),silicon, germanium, graphite, ceramics, silicon, semiconductor, highrefractive index dielectrics, crystals, gels, polymers, or films (e.g.,films of gold, silver, aluminum, or diamond).

In some embodiments, a surface can be magnetic or paramagnetic bead(e.g., magnetic or paramagnetic nanoparticles or microparticles). Insome embodiments, paramagnetic microparticles can be paramagnetic beadsattached with streptavidin (e.g., Dynabeads™ M-270 from Invitrogen,Carlsbad, Calif.). Particles can have an iron core, or comprise ahydrogel or agarose (e.g., Sepharose™).

In some embodiments, the surface can be attached with a plurality of afirst primer. A surface can be coated with an acrylamide, carboxylic oramine compound for attaching a nucleic acid (e.g., a first primer). Insome embodiments, an amino-modified nucleic acid (e.g., primer) can beattached to a surface that is coated with a carboxylic acid. In someembodiments, an amino-modified nucleic acid can be reacted with EDC (orEDAC) for attachment to a carboxylic acid coated surface (with orwithout NHS). A first primer can be immobilized to an acrylamidecompound coating on a surface. Particles can be coated with anavidin-like compound (e.g., streptavidin) for binding biotinylatednucleic acids.

In some embodiments, the surface comprises the surface of a bead. Insome embodiments, a bead comprises a polymer material. For example, abead comprises a gel, hydrogel or acrylamide polymers. A bead can beporous. Particles can have cavitation or pores, or can includethree-dimensional scaffolds. In some embodiments, particles can be IonSphere™ particles.

In some embodiments, the disclosed methods (as well as relatedcompositions, systems and kits) include immobilizing one or more nucleicacid templates onto one or more supports. Nucleic acids may beimmobilized on the solid support by any method including but not limitedto physical adsorption, by ionic or covalent bond formation, orcombinations thereof. A solid support may include a polymeric, a glass,or a metallic material. Examples of solid supports include a membrane, aplanar surface, a microtiter plate, a bead, a filter, a test strip, aslide, a cover slip, and a test tube. A support includes any solid phasematerial upon which a oligomer is synthesized, attached, ligated orotherwise immobilized. A support can optionally comprise a “resin”,“phase”, “surface” and “support”. A support may be composed of organicpolymers such as polystyrene, polyethylene, polypropylene,polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well asco-polymers and grafts thereof. A support may also be inorganic, such asglass, silica, controlled-pore-glass (CPG), or reverse-phase silica. Theconfiguration of a support may be in the form of beads, spheres,particles, granules, a gel, or a surface. Surfaces may be planar,substantially planar, or non-planar. Supports may be porous ornon-porous, and may have swelling or non-swelling characteristics. Asupport can be shaped to comprise one or more wells, depressions orother containers, vessels, features or locations. A plurality ofsupports may be configured in an array at various locations. A supportis optionally addressable (e.g., for robotic delivery of reagents), orby detection means including scanning by laser illumination and confocalor deflective light gathering. An amplification support (e.g., a bead)can be placed within or on another support (e.g., within a well of asecond support).

In an embodiment the solid support is a “microparticle,” “bead”“microbead”, etc., (optionally but not necessarily spherical in shape)having a smallest cross-sectional length (e.g., diameter) of 50 micronsor less, preferably 10 microns or less, 3 microns or less, approximately1 micron or less, approximately 0.5 microns or less, e.g., approximately0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer,about 1-10 nanometer, about 10-100 nanometers, or about 100-500nanometers). Microparticles (e.g., Dynabeads from Dynal, Oslo, Norway)may be made of a variety of inorganic or organic materials including,but not limited to, glass (e.g., controlled pore glass), silica,zirconia, cross-linked polystyrene, polyacrylate, polymehtymethacrylate,titanium dioxide, latex, polystyrene, etc. Magnetization can facilitatecollection and concentration of the microparticle-attached reagents(e.g., polynucleotides or ligases) after amplification, and can alsofacilitate additional steps (e.g., washes, reagent removal, etc.). Incertain embodiments of the invention a population of microparticleshaving different shapes sizes and/or colors can be used. Themicroparticles can optionally be encoded, e.g., with quantum dots suchthat each microparticle can be individually or uniquely identified.

In some embodiments, a bead surface can be functionalized for attachinga plurality of a first primer. In some embodiments, a bead can be anysize that can fit into a reaction chamber. For example, one bead can fitin a reaction chamber. In some embodiments more than one bead can fit ina reaction chamber. In some embodiments, the smallest cross-sectionallength of a bead (e.g., diameter) can be about 50 microns or less, orabout 10 microns or less, or about 3 microns or less, approximately 1micron or less, approximately 0.5 microns or less, e.g., approximately0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer,about 1-10 nanometer, about 10-100 nanometers, or about 100-500nanometers). In some embodiments, a bead can be attached with aplurality of one or more different primer sequences. In someembodiments, a bead can be attached with a plurality of one primersequence, or can be attached a plurality of two or more different primersequences. In some embodiments, a bead can be attached with a pluralityof at least 1,000 primers, or about 1,000-10,000 primers, or about,10,000-50,000 primers, or about 50,000-75,000 primers, or about75,000-100,000 primers, or more.

In some embodiments, nucleotides can be compatible for use in any typeof sequencing platform including chemical degradation,chain-termination, sequence-by-synthesis, pyrophosphate, massivelyparallel, ion-sensitive, and single molecule platforms.

In some embodiments, nucleotides can be used in any nucleic acidsequencing workflow, including sequencing by oligonucleotide probeligation and detection (e.g., SOLiD™ from Life Technologies, WO2006/084131), probe-anchor ligation sequencing (e.g., Complete Genomics™or Polonator™), sequencing-by-synthesis (e.g., Genetic Analyzer andHiSeq™, from 11lumina), pyrophosphate sequencing (e.g., Genome SequencerFLX from 454 Life Sciences), ion-sensitive sequencing (e.g., PersonalGenome Machine (PGM™) and Ion Proton™ Sequencer, both from Ion TorrentSystems, Inc.), and single molecule sequencing platforms (e.g.,HeliScope™ from Helicos™).

In some embodiments, the disclosure relates generally to compositions,as well as related methods, systems, kits and apparatuses, fornucleotide incorporation reactions comprising nucleic acid sequencingmethods that detect one or more byproducts of nucleotide incorporation.The detection of polymerase extension by detecting physicochemicalbyproducts of the extension reaction, can include polyphosphate,pyrophosphate, hydrogen ion, charge transfer, heat, and the like, asdisclosed, for example, in U.S. Pat. No. 7,948,015 to Rothberg et al.;and Rothberg et al, U.S. Patent Publication No. 2009/0026082, herebyincorporated by reference in their entireties. Other examples of methodsof detecting polymerase-based extension can be found, for example, inPourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006);Purushothaman et al., IEEE ISCAS, IV-169-172; Anderson et al, Sensorsand Actuators B Chem., 129: 79-86 (2008); Sakata et al., Angew. Chem.118:2283-2286 (2006); Esfandyapour et al., U.S. Patent Publication No.2008/01666727; and Sakurai et al., Anal. Chem. 64: 1996-1997 (1992).

Reactions involving the generation and detection of ions are widelyperformed. The use of direct ion detection methods to monitor theprogress of such reactions can simplify many current biological assays.For example, template-dependent nucleic acid synthesis by a polymerasecan be monitored by detecting hydrogen ions that are generated asnatural byproducts of nucleotide incorporations catalyzed by thepolymerase. Ion-sensitive sequencing (also referred to as “pH-based” or“ion-based” nucleic acid sequencing) exploits the direct detection ofionic byproducts, such as hydrogen ions, that are produced as abyproduct of nucleotide incorporation. In one exemplary system forion-based sequencing, the nucleic acid to be sequenced can be capturedin a microwell, and nucleotides can be flowed across the well, one at atime, under nucleotide incorporation conditions. The polymeraseincorporates the appropriate nucleotide into the growing strand, and thehydrogen ion that is released can change the pH in the solution, whichcan be detected by an ion sensor that is coupled with the well. Thistechnique does not require labeling of the nucleotides or expensiveoptical components, and allows for far more rapid completion ofsequencing runs. Examples of such ion-based nucleic acid sequencingmethods and platforms include the Ion Torrent PGM™ or Proton™ sequencer(Ion Torrent™ Systems, Life Technologies Corporation).

In some embodiments, target polynucleotides produced using the methods,systems and kits of the present teachings can be used as a substrate fora biological or chemical reaction that is detected and/or monitored by asensor including a field-effect transistor (FET). In various embodimentsthe FET is a chemFET or an ISFET. A “chemFET” or chemical field-effecttransistor, is a type of field effect transistor that acts as a chemicalsensor. It is the structural analog of a MOSFET transistor, where thecharge on the gate electrode is applied by a chemical process. An“ISFET” or ion-sensitive field-effect transistor, is used for measuringion concentrations in solution; when the ion concentration (such as H+)changes, the current through the transistor will change accordingly. Adetailed theory of operation of an ISFET is given in “Thirty years ofISFETOLOGY: what happened in the past 30 years and what may happen inthe next 30 years,” P. Bergveld, Sens. Actuators, 88 (2003), pp. 1-20.

In some embodiments, the FET may be a FET array. As used herein, an“array” is a planar arrangement of elements such as sensors or wells.The array may be one or two dimensional. A one dimensional array can bean array having one column (or row) of elements in the first dimensionand a plurality of columns (or rows) in the second dimension. The numberof columns (or rows) in the first and second dimensions may or may notbe the same. The FET or array can comprise 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷or more FETs.

In some embodiments, one or more microfluidic structures can befabricated above the FET sensor array to provide for containment and/orconfinement of a biological or chemical reaction. For example, in oneimplementation, the microfluidic structure(s) can be configured as oneor more wells (or microwells, or reaction chambers, or reaction wells,as the terms are used interchangeably herein) disposed above one or moresensors of the array, such that the one or more sensors over which agiven well is disposed detect and measure analyte presence, level,and/or concentration in the given well. In some embodiments, there canbe a 1:1 correspondence of FET sensors and reaction wells.

Microwells or reaction chambers are typically hollows or wells havingwell-defined shapes and volumes which can be manufactured into asubstrate and can be fabricated using conventional microfabricationtechniques, e.g. as disclosed in the following references: Doering andNishi, Editors, Handbook of Semiconductor Manufacturing Technology,Second Edition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMSand Medical Microdevices (SPIE Publications, 2006); Elwenspoek et al,Silicon Micromachining (Cambridge University Press, 2004); and the like.Examples of configurations (e.g. spacing, shape and volumes) ofmicrowells or reaction chambers are disclosed in Rothberg et al, U.S.patent publication 2009/0127589; Rothberg et al, U.K. patent applicationGB24611127.

In some embodiments, the biological or chemical reaction can beperformed in a solution or a reaction chamber that is in contact with,operatively coupled, or capacitively coupled to a FET such as a chemFETor an ISFET. The FET (or chemFET or ISFET) and/or reaction chamber canbe an array of FETs or reaction chambers, respectively.

In some embodiments, a biological or chemical reaction can be carriedout in a two-dimensional array of reaction chambers, wherein eachreaction chamber can be coupled to a FET, and each reaction chamber isno greater than 10 μm³ (i.e., 1 pL) in volume. In some embodiments eachreaction chamber is no greater than 0.34 pL, 0.096 pL or even 0.012 pLin volume. A reaction chamber can optionally be no greater than 2, 5,10, 15, 22, 32, 42, 52, 62, 72, 82, 92, or 102 square microns incross-sectional area at the top. Preferably, the array has at least 10²,10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or more reaction chambers. In someembodiments, at least one of the reaction chambers is operativelycoupled to at least one of the FETs.

FET arrays as used in various embodiments according to the disclosurecan be fabricated according to conventional CMOS fabricationstechniques, as well as modified CMOS fabrication techniques and othersemiconductor fabrication techniques beyond those conventionallyemployed in CMOS fabrication. Additionally, various lithographytechniques can be employed as part of an array fabrication process.

Exemplary FET arrays suitable for use in the disclosed methods, as wellas microwells and attendant fluidics, and methods for manufacturingthem, are disclosed, for example, in U.S. Patent Publication No.2010/0301398; U.S. Patent Publication No. 2010/0300895; U.S. PatentPublication No. 2010/0300559 (now U.S. Pat. No. 8,546,128); U.S. PatentPublication No. 2010/0197507 (now U.S. Pat. No. 8,306,757); U.S. PatentPublication No. 2010/0137143; U.S. Patent Publication No. 2009/0127589(now U.S. Pat. No. 7,948,015); and U.S. Patent Publication No.2009/0026082 (now U.S. Pat. No. 8,262,900), which are incorporated byreference in their entireties.

In one aspect, the disclosed compositions, methods, systems, apparatusesand kits can be used for carrying out label-free nucleic acidsequencing, and in particular, ion-based nucleic acid sequencing. Theconcept of label-free detection of nucleotide incorporation has beendescribed in the literature, including the following references that areincorporated by reference: Rothberg et al, U.S. patent publication2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470(2006). Briefly, in nucleic acid sequencing applications, nucleotideincorporations are determined by measuring natural byproducts ofpolymerase-catalyzed extension reactions, including hydrogen ions,polyphosphates, PPi, and Pi (e.g., in the presence of phosphatase orpyrophosphatase). Examples of such ion-based nucleic acid sequencingmethods and platforms include the Ion Torrent PGM™ or Proton™ sequencer(Ion Torrent™ Systems, Life Technologies Corporation).

In some embodiments, the present teachings provide nucleotides employedin a nucleic acid sequencing method. In one exemplary embodiment, thedisclosure relates generally to a method for obtaining sequenceinformation from template polynucleotides, comprising: performingtemplate-dependent nucleic acid synthesis using any one, or acombination of any, of the nucleotides described herein.

In some embodiments, the template-dependent synthesis includesincorporating one or more nucleotides in a template-dependent fashioninto a newly synthesized nucleic acid strand.

Optionally, the methods can further include producing one or more ionicbyproducts of such nucleotide incorporation.

In some embodiments, the methods can further include detecting theincorporation of the one or more nucleotides into the sequencing primer.Optionally, the detecting can include detecting the release of hydrogenions.

In some embodiments, the disclosure relates generally to a method forsequencing a nucleic acid, comprising: (a) disposing templatepolynucleotides into a plurality of reaction chambers, wherein one ormore of the reaction chambers are in contact with at least one fieldeffect transistor (FET). Optionally, the method further includescontacting template polynucleotides, which are disposed into one of thereaction chambers, with a polymerase thereby synthesizing a new nucleicacid strand by sequentially incorporating one or more nucleotides into anucleic acid molecule. Optionally, the method further includesgenerating one or more hydrogen ions as a byproduct of such nucleotideincorporation. Optionally, the method further includes detecting theincorporation of the one or more nucleotides by detecting the generationof the one or more hydrogen ions using the FET.

In some embodiments, the detecting includes detecting a change involtage and/or current at the at least one FET within the array inresponse to the generation of the one or more hydrogen ions.

In some embodiments, the FET can be selected from the group consistingof: ion-sensitive FET (isFET) and chemically-sensitive FET (chemFET).

One exemplary system involving sequencing via detection of ionicbyproducts of nucleotide incorporation is the Ion Torrent PGM™ orProton™ sequencer (Life Technologies), which is an ion-based sequencingsystem that sequences nucleic acid templates by detecting hydrogen ionsproduced as a byproduct of nucleotide incorporation. Typically, hydrogenions are released as byproducts of nucleotide incorporations occurringduring template-dependent nucleic acid synthesis by a polymerase. TheIon Torrent PGM™ or Proton™ sequencer detects the nucleotideincorporations by detecting the hydrogen ion byproducts of thenucleotide incorporations. The Ion Torrent PGM™ or Proton™ sequencer caninclude a plurality of nucleic acid templates to be sequenced, eachtemplate disposed within a respective sequencing reaction well in anarray. The wells of the array can each be coupled to at least one ionsensor that can detect the release of H⁺ ions or changes in solution pHproduced as a byproduct of nucleotide incorporation. The ion sensorcomprises a field effect transistor (FET) coupled to an ion-sensitivedetection layer that can sense the presence of H⁺ ions or changes insolution pH. The ion sensor can provide output signals indicative ofnucleotide incorporation which can be represented as voltage changeswhose magnitude correlates with the H⁺ ion concentration in a respectivewell or reaction chamber. Different nucleotide types can be flowedserially into the reaction chamber, and can be incorporated by thepolymerase into an extending primer (or polymerization site) in an orderdetermined by the sequence of the template. Each nucleotideincorporation can be accompanied by the release of H⁺ ions in thereaction well, along with a concomitant change in the localized pH. Therelease of H⁺ ions can be registered by the FET of the sensor, whichproduces signals indicating the occurrence of the nucleotideincorporation. Nucleotides that are not incorporated during a particularnucleotide flow may not produce signals. The amplitude of the signalsfrom the FET can also be correlated with the number of nucleotides of aparticular type incorporated into the extending nucleic acid moleculethereby permitting homopolymer regions to be resolved. Thus, during arun of the sequencer multiple nucleotide flows into the reaction chamberalong with incorporation monitoring across a multiplicity of wells orreaction chambers can permit the instrument to resolve the sequence ofmany nucleic acid templates simultaneously. Further details regardingthe compositions, design and operation of the Ion Torrent PGM™ orProton™ sequencer can be found, for example, in U.S. Patent PublicationNo. 2009/0026082 (now U.S. Pat. No. 8,262,900); U.S. Patent PublicationNo. 2010/0137143; and U.S. Patent Publication No. 2010/0282617 (now U.S.Pat. No. 8,349,167), all of which applications are incorporated byreference herein in their entireties.

In various exemplary embodiments, the methods, systems, and computerreadable media described herein may advantageously be used to processand/or analyze data and signals obtained from electronic orcharged-based nucleic acid sequencing. In electronic or charged-basedsequencing (such as, pH-based sequencing), a nucleotide incorporationevent may be determined by detecting ions (e.g., hydrogen ions) that aregenerated as natural by-products of polymerase-catalyzed nucleotideextension reactions. This may be used to sequence a sample or templatenucleic acid, which may be a fragment of a nucleic acid sequence ofinterest, for example, and which may be directly or indirectly attachedas a clonal population to a solid support, such as a particle,microparticle, bead, etc. The sample or template nucleic acid may beoperably associated to a primer and polymerase and may be subjected torepeated cycles or “flows” of nucleotide addition (which may be referredto herein as “nucleotide flows” from which nucleotide incorporations mayresult) and washing. The primer may be annealed to the sample ortemplate so that the primer's 3′ end can be extended by a polymerasewhenever nucleotides complementary to the next base in the template areadded. Then, based on the known sequence of nucleotide flows and onmeasured output signals of the chemical sensors indicative of ionconcentration during each nucleotide flow, the identity of the type,sequence and number of nucleotide(s) associated with a sample nucleicacid present in a reaction region coupled to a chemical sensor can bedetermined.

In a typical embodiment of ion-based nucleic acid sequencing, nucleotideincorporations can be detected by detecting the presence and/orconcentration of hydrogen ions generated by polymerase-catalyzedextension reactions. In one embodiment, templates, optionally pre-boundto a sequencing primer and/or a polymerase, can be loaded into reactionchambers (such as the microwells disclosed in Rothberg et al, citedherein), after which repeated cycles of nucleotide addition and washingcan be carried out. In some embodiments, such templates can be attachedas clonal populations to a solid support, such as particles, bead, orthe like, and said clonal populations are loaded into reaction chambers.

In another embodiment, the templates, optionally bound to a polymerase,are distributed, deposited or positioned to different sites of thearray. The sites of the array include primers and the methods caninclude hybridizing different templates to the primers within differentsites.

In each addition step of the cycle, the polymerase can extend the primerby incorporating added nucleotide only if the next base in the templateis the complement of the added nucleotide. If there is one complementarybase, there is one incorporation, if two, there are two incorporations,if three, there are three incorporations, and so on. With each suchincorporation there is a hydrogen ion released, and collectively apopulation of templates releasing hydrogen ions changes the local pH ofthe reaction chamber. The production of hydrogen ions is monotonicallyrelated to the number of contiguous complementary bases in the template(as well as the total number of template molecules with primer andpolymerase that participate in an extension reaction). Thus, when thereare a number of contiguous identical complementary bases in the template(i.e. a homopolymer region), the number of hydrogen ions generated, andtherefore the magnitude of the local pH change, can be proportional tothe number of contiguous identical complementary bases. If the next basein the template is not complementary to the added nucleotide, then noincorporation occurs and no hydrogen ion is released. In someembodiments, after each step of adding a nucleotide, an additional stepcan be performed, in which an unbuffered wash solution at apredetermined pH is used to remove the nucleotide of the previous stepin order to prevent misincorporations in later cycles. In someembodiments, the after each step of adding a nucleotide, an additionalstep can be performed wherein the reaction chambers are treated with anucleotide-destroying agent, such as apyrase, to eliminate any residualnucleotides remaining in the chamber, which may result in spuriousextensions in subsequent cycles.

In one exemplary embodiment, different kinds of nucleotides are addedsequentially to the reaction chambers, so that each reaction can beexposed to the different nucleotides one at a time. For example,nucleotides can be added in the following sequence: dATP, dCTP, dGTP,dTTP, dATP, dCTP, dGTP, dTTP, and so on; with each exposure followed bya wash step. The cycles may be repeated for 50 times, 100 times, 200times, 300 times, 400 times, 500 times, 750 times, or more, depending onthe length of sequence information desired.

In some embodiments, sequencing can be performed according to the userprotocols supplied with the PGM™ or Proton™ sequencer. Example 3provides one exemplary protocol for ion-based sequencing using the IonTorrent PGM™ sequencer (Ion Torrent™ Systems, Life Technologies,Calif.).

In some embodiments, the disclosure relates generally to methods forsequencing a population of template polynucleotides, comprising: (a)generating a plurality of amplicons by clonally amplifying a pluralityof template polynucleotides onto a plurality of surfaces, wherein theamplifying is performed within a single continuous phase of a reactionmixture and wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% of the resulting amplicons are substantially monoclonal innature. In some embodiments, a sufficient number of substantiallymonoclonal amplicons are produced in a single amplification reaction togenerate at least 100 MB, 200MB, 300 MB, 400 MB, 500MB, 750 MB, 1GB or 2GB of AQ20 sequencing reads on an Ion Torrent PGM™ 314, 316 or 318sequencer. The term “AQ20 and its variants, as used herein, refers to aparticular method of measuring sequencing accuracy in the Ion TorrentPGM™ sequencer. Accuracy can be measured in terms of the Phred-like Qscore, which measures accuracy on logarithmic scale that: Q10=90%,Q20=99%, Q30=99.9%, Q40=99.99%, and Q50=99.999%. For example, in aparticular sequencing reaction, accuracy metrics can be calculatedeither through prediction algorithms or through actual alignment to aknown reference genome. Predicted quality scores (“Q scores”) can bederived from algorithms that look at the inherent properties of theinput signal and make fairly accurate estimates regarding if a givensingle base included in the sequencing “read” will align. In someembodiments, such predicted quality scores can be useful to filter andremove lower quality reads prior to downstream alignment. In someembodiments, the accuracy can be reported in terms of a Phred-like Qscore that measures accuracy on logarithmic scale such that: Q10=90%,Q17=98%, Q20=99%, Q30=99.9%, Q40=99.99%, and Q50=99.999%. In someembodiments, the data obtained from a given polymerase reaction can befiltered to measure only polymerase reads measuring “N” nucleotides orlonger and having a Q score that passes a certain threshold, e.g., Q10,Q17, Q100 (referred to herein as the “NQ17” score). For example, the100Q20 score can indicate the number of reads obtained from a givenreaction that are at least 100 nucleotides in length and have Q scoresof Q20 (99%) or greater. Similarly, the 200Q20 score can indicate thenumber of reads that are at least 200 nucleotides in length and have Qscores of Q20 (99%) or greater.

In some embodiments, accuracy can also be calculated based on properalignment using a reference genomic sequence, referred to herein as the“raw” accuracy. This is single pass accuracy, involving measurement ofthe “true” per base error associated with a single read, as opposed toconsensus accuracy, which measures the error rate from the consensussequence which is the result of multiple reads. Raw accuracymeasurements can be reported in terms of “AQ” scores (for alignedquality). In some embodiments, the data obtained from a given polymerasereaction can be filtered to measure only polymerase reads measuring “N”nucleotides or longer having a AQ score that passes a certain threshold,e.g., AQ10, AQ17, AQ100 (referred to herein as the “NAQ17” score). Forexample, the 100AQ20 score can indicate the number of reads obtainedfrom a given polymerase reaction that are at least 100 nucleotides inlength and have AQ scores of AQ20 (99%) or greater. Similarly, the200AQ20 score can indicate the number of reads that are at least 200nucleotides in length and have AQ scores of AQ20 (99%) or greater.

Embodiments of the present teachings can be further understood in lightof the following examples, which should not be construed as limiting thescope of the present teachings in any way.

EXAMPLE 1

Polymerase Incorporation of γ-S-TTP: Effect of Alkaline PhosphatasePre-Treatment:

This experiment was conducted to demonstrate that γ-S-TTP is resistantto degradation by calf intestinal phosphatase.

Two solutions were prepared as follows in 20 mM Tris HCl, pH 7.5, 20 mMNaCl, 10 mM MgCl₂: (A) 80 μM of TTP, and (B) 200 μM of γ-S-TTP.

To both solution was added 1.5 μL of 10 u/μL of calf intestinalphosphatase (CIP), and the solutions were incubated at 37° C. for 20min. The phosphatase was then removed by passing the solutions through a10,000 molecular weight cut-off ultrafiltration membrane. The resultingsolutions were then used for the polymerase extension reactions.

For the polymerase extension assays, a fluorescein-labeledoligonucleotide substrate was designed, and used in an assay to detectnucleotide incorporation. The labeled oligonucleotide is a hairpin-typeoligonucleotide with a T-fluorescein residue located at the thirdposition from the 3′ end. The 5′ end of the labeled oligonucleotide is asingle stranded sequence 3′-AGGGGGG-5′. Thus, the first nucleotide to beinserted by a polymerase onto the 3′ end of the substrate is a TTP. The5′ and 3′ ends of the hairpin oligonucleotide is shown below. Afluorescein is attached to the T residue, which is underlined andbolded:

-   TGC 3′-   ACGAGGGGGG 5′ (SEQ ID NO:83)

Without wishing to be bound by theory, it is postulated that when thethird base from the 3′ end is labeled with a fluorphore (in this casethe labeled nucleotide is a T), and when the 3′ terminal base is acytosine or guanosine, then the fluorphore is in a stacked position withthe 3′ terminal base, resulting in quenching the fluorescence from thefluorphore. When a thymidine is incorporated at the terminal 3′ end(e.g., via polymerase-mediated nucleotide incorporation), the fluorphoreis un-quenched. This assay can be performed with the hairpin moleculedescribed herein, or with two annealed single-strands that mimic thelinear ends of this hairpin molecule.

A solution was prepared in the same buffer as above containing 250 nM ofthe hairpin oligonucleotide, 16 μM of a short oligonucleotide(5′-CCCCCC-3′, acting as a signal amplifier), and 2 nM of Bstpolymerase. Then 50 μL aliquots of this solution were placed inindividual wells of a microtiter plate. In separate wells of the sameplate were placed 50 μL aliquots of the CIP-treated solutions fromabove, as well as identical solutions that had not been treated withCIP. The plate was equilibrated at 37° C. in a fluorescence microplatereader, and then the dNTP solutions were mixed with the solutions ofpolymerase and fluorogenic hairpin oligonucleotide. The polymerasecatalyzed incorporation reactions were followed by recording thefluorescence intensity changes using 490 nm for excitation and 525 nmfor the emission. When a polymerase reaction occurred, an increase inthe fluorescence intensity over time was detected (see FIG. 4). Therewas no change in fluorescence when no nucleotide incorporation reactionoccurred (see FIG. 4).

FIG. 4 shows that no incorporation reaction takes place with aCIP-treated TTP. On the other hand when a γ-S-TTP was used, polymerasereactions can be detected with both CIP treated and non-treatedsolutions. This demonstrates that the γ-S-TTP is resistant to thealkaline phosphatase activity. A treatment of synthetically preparedγ-S-TTP with CIP can be used to remove any traces of contaminating dTTPthat may be present in the preparation of γ-S-TTP.

TABLE 1 Some Typical pKa Values For Free Amino Acids In Solution AminoAcid α-carboxylic acid α-amino Side chain Alanine 2.35 9.87 Arginine2.01 9.04 12.48 Asparagine 2.02 8.80 Aspartic Acid 2.10 9.82 3.86Cysteine 2.05 10.25 8.00 Glutamic Acid 2.10 9.47 4.07 Glutamine 2.179.13 Glycine 2.35 9.78 Histidine 1.77 9.18 6.10 Isoleucine 2.32 9.76Leucine 2.33 9.74 Lysine 2.18 8.95 10.53 Methionine 2.28 9.21Phenylalanine 2.58 9.24 Proline 2.00 10.60 Serine 2.21 9.15 Threonine2.09 9.10 Tryptophan 2.38 9.39 Tyrosine 2.20 9.11 10.07 Valine 2.29 9.72

TABLE 2 Candidate amino acid residues for modification in Bst DNApolymerase (including pKa values for amino acid residues calculatedusing PropKa) Table 2 Amino Acid Residue pKa GLU-277 4.64 GLU-372 4.7GLU-15 4.71 GLU-206 4.71 GLU-426 4.71 GLU-493 4.71 GLU-456 4.76 GLU-1314.78 GLU-349 4.78 GLU-446 4.85 GLU-522 4.85 GLU-558 4.85 GLU-26 4.92GLU-294 4.92 GLU-363 5.08 HIS-534 5.12 HIS-572 6.17 S HIS-473 6.29 BHIS-46 6.43 S HIS-273 6.51 S LYS-510 7.29 B N+ 7.86 S TYR-477 7.98 BLYS-73 8.55 CYS-550 8.87 CYS-93 9.57

TABLE 3 Candidate amino acid residues for modification in Bst DNApolymerase (including pKa values for amino acid residues calculatedusing H++) Table 3 Amino Acid Residue pKa GLU-461 4.533 GLU-206 4.555ASP-113 4.57 HIS-273 4.662 HIS-534 4.747 GLU-277 4.806 GLU-456 4.902GLU-544 4.972 GLU-54 5.037 GLU-30 5.149 GLU-349 5.158 GLU-522 5.221HIS-151 5.26 GLU-558 5.361 GLU-220 5.423 GLU-446 5.911 S NTA 6.465 SHIS-46 7.653 S HIS-572 8.056 S HIS-308 8.333 HIS-473 8.397 LYS-411 9.494LYS-543 9.72 LYS-287 9.815 TYR-419 10.041 LYS-253 10.077

TABLE 4 Candidate amino acids for modification in E. coli SSB Table 4Amino Acid Residue pKa HIS-55 3.968 ASP-90 4.251 ASP-17 4.298 ASP-424.449 GLU-50 4.738 GLU-65 4.856 GLU-47 4.878 GLU-19 4.896 GLU-53 5.046ASP-95 5.143 GLU-69 5.214 GLU-38 5.606 NTALA-1 5.851 GLU-80 5.898 LYS-77.276 LYS-49 8.791 GLU-100 9.033 ARG-3 9.069 LYS-87 9.453 LYS-62 9.754LYS-43 10.399 TYR-22 10.427 TYR-97 10.483 TYR-70 10.619 LYS-73 10.898ARG-56 11.169 ARG-96 11.176 ARG-86 11.257 ARG-84 11.296 ARG-41 11.381ARG-115 11.804 ARG-21 12.035 ARG-72 12.671 TYR-78 16.412

TABLE 5 Candidate amino acids for substitution in Therminator ™ DNApolymerase Table 5 Amino Acid pKa pKa Residue (calc) (model) ASP 4 7.85ASP 6 5.95 ASP 31 2.11 ASP 44 2.53 ASP 45 3.60 ASP 50 2.82 ASP 92 4.14ASP 98 3.43 ASP 108 4.05 3.80 ASP 113 3.10 3.80 ASP 123 4.09 3.80 ASP132 3.64 3.80 ASP 164 1.56 3.80 ASP 177 3.87 3.80 ASP 182 3.79 3.80 ASP202 3.47 3.80 ASP 204 2.82 3.80 ASP 212 2.14 3.80 ASP 215 7.23 3.80 ASP235 2.59 3.80 ASP 246 3.29 3.80 ASP 259 6.50 3.80 ASP 315 5.97 3.80 ASP343 3.69 3.80 ASP 373 3.18 3.80 ASP 398 4.52 3.80 ASP 404 6.10 3.80 ASP421 4.42 3.80 ASP 432 2.60 3.80 ASP 444 3.20 3.80 ASP 455 3.97 3.80 ASP472 3.03 3.80 ASP 480 3.21 3.80 ASP 540 3.92 3.80 ASP 542 4.89 3.80 ASP552 2.69 3.80 ASP 598 3.40 3.80 ASP 614 3.89 3.80 ASP 635 2.78 3.80 ASP712 3.83 3.80 ASP 718 3.80 3.80 GLU 10 4.05 4.50 GLU 22 4.12 4.50 GLU 253.60 4.50 GLU 29 4.26 4.50 GLU 35 2.57 4.50 GLU 49 3.85 4.50 GLU 69 4.564.50 GLU 81 4.38 4.50 GLU 111 4.60 4.50 GLU 130 4.51 4.50 GLU 133 3.914.50 GLU 134 4.91 4.50 GLU 148 5.27 4.50 GLU 150 5.08 4.50 GLU 151 4.104.50 GLU 167 4.42 4.50 GLU 187 4.70 4.50 GLU 189 3.57 4.50 GLU 200 4.184.50 GLU 224 4.29 4.50 GLU 225 4.55 4.50 GLU 238 4.49 4.50 GLU 251 4.604.50 GLU 276 4.43 4.50 GLU 280 4.75 4.50 GLU 288 3.94 4.50 GLU 293 4.714.50 GLU 294 3.67 4.50 GLU 300 4.26 4.50 GLU 303 4.59 4.50 GLU 306 4.724.50 GLU 314 5.00 4.50 GLU 321 4.64 4.50 GLU 325 4.87 4.50 GLU 330 7.314.50 GLU 354 5.67 4.50 GLU 366 6.07 4.50 GLU 374 4.64 4.50 GLU 376 3.894.50 GLU 391 4.65 4.50 GLU 393 3.42 4.50 GLU 426 4.46 4.50 GLU 430 4.754.50 GLU 436 4.54 4.50 GLU 458 4.49 4.50 GLU 459 3.84 4.50 GLU 475 4.114.50 GLU 508 4.65 4.50 GLU 511 3.78 4.50 GLU 519 4.91 4.50 GLU 522 4.094.50 GLU 527 2.97 4.50 GLU 529 4.67 4.50 GLU 530 4.53 4.50 GLU 554 4.844.50 GLU 562 4.46 4.50 GLU 576 5.03 4.50 GLU 578 3.64 4.50 GLU 580 4.994.50 GLU 599 5.35 4.50 GLU 600 6.04 4.50 GLU 609 5.62 4.50 GLU 617 4.454.50 GLU 621 4.96 4.50 GLU 628 4.02 4.50 GLU 637 5.22 4.50 GLU 638 4.754.50 GLU 645 4.50 4.50 GLU 664 4.36 4.50 GLU 719 4.28 4.50 GLU 730 4.724.50 GLU 734 4.98 4.50 GLU 742 3.65 4.50 C− 750 3.25 3.20 HIS 59 6.136.50 HIS 89 4.69 6.50 HIS 103 7.00 6.50 HIS 147 7.17 6.50 HIS 257 4.016.50 HIS 416 5.54 6.50 HIS 439 6.77 6.50 HIS 545 2.90 6.50 HIS 633 6.966.50 HIS 663 5.84 6.50 HIS 679 6.64 6.50 CYS 223 11.84 9.00 CYS 42899.99 99.99 CYS 442 99.99 99.99 CYS 506 99.99 99.99 CYS 509 99.99 99.99TYR 7 10.59 10.00 TYR 30 10.26 10.00 TYR 37 17.23 10.00 TYR 39 14.2010.00 TYR 86 10.20 10.00 TYR 110 11.95 10.00 TYR 112 10.49 10.00 TYR 12013.12 10.00 TYR 146 11.21 10.00 TYR 162 11.82 10.00 TYR 180 11.47 10.00TYR 209 13.47 10.00 TYR 218 11.91 10.00 TYR 261 10.23 10.00 TYR 273 9.7710.00 TYR 279 11.96 10.00 TYR 291 10.52 10.00 TYR 311 14.21 10.00 TYR320 10.89 10.00 TYR 362 11.49 10.00 TYR 384 11.17 10.00 TYR 388 12.2310.00 TYR 402 14.32 10.00 TYR 409 14.74 10.00 TYR 431 10.05 10.00 TYR481 10.52 10.00 TYR 494 12.59 10.00 TYR 496 16.00 10.00 TYR 497 14.4010.00 TYR 499 11.49 10.00 TYR 505 11.27 10.00 TYR 520 11.38 10.00 TYR538 13.52 10.00 TYR 566 11.47 10.00 TYR 579 11.62 10.00 TYR 583 11.5510.00 TYR 594 12.23 10.00 TYR 701 14.24 10.00 TYR 731 10.99 10.00 TYR732 11.88 10.00 TYR 750 10.81 10.00 LYS 13 10.18 10.50 LYS 20 11.1010.50 LYS 21 10.56 10.50 LYS 27 11.24 10.50 LYS 43 10.25 10.50 LYS 5211.08 10.50 LYS 53 10.63 10.50 LYS 57 10.28 10.50 LYS 64 10.26 10.50 LYS66 10.51 10.50 LYS 70 11.41 10.50 LYS 73 9.02 10.50 LYS 74 10.47 10.50LYS 84 10.64 10.50 LYS 118 10.50 10.50 LYS 124 10.20 10.50 LYS 174 10.0110.50 LYS 175 10.42 10.50 LYS 188 10.15 10.50 LYS 192 11.36 10.50 LYS201 12.01 10.50 LYS 220 10.65 10.50 LYS 221 10.80 10.50 LYS 229 10.5410.50 LYS 240 10.09 10.50 LYS 253 11.39 10.50 LYS 285 10.22 10.50 LYS287 11.74 10.50 LYS 289 9.16 10.50 LYS 317 10.31 10.50 LYS 360 8.9110.50 LYS 363 10.27 10.50 LYS 371 9.91 10.50 LYS 390 10.38 10.50 LYS 42910.64 10.50 LYS 440 10.41 10.50 LYS 443 10.92 10.50 LYS 462 11.52 10.50LYS 464 8.83 10.50 LYS 466 10.69 10.50 LYS 468 10.07 10.50 LYS 476 10.2610.50 LYS 477 10.91 10.50 LYS 487 10.75 10.50 LYS 501 10.22 10.50 LYS507 10.64 10.50 LYS 531 11.77 10.50 LYS 535 10.87 10.50 LYS 557 10.5110.50 LYS 558 10.76 10.50 LYS 559 10.31 10.50 LYS 561 10.04 10.50 LYS565 10.25 10.50 LYS 591 10.45 10.50 LYS 592 10.20 10.50 LYS 593 9.7410.50 LYS 602 10.54 10.50 LYS 620 10.49 10.50 LYS 632 10.36 10.50 LYS644 10.24 10.50 LYS 684 10.40 10.50 LYS 692 10.25 10.50 LYS 705 9.5710.50 LYS 746 11.49 10.50 ARG 17 12.25 12.50 ARG 32 13.03 12.50 ARG 5812.29 12.50 ARG 67 14.10 12.50 ARG 78 12.36 12.50 ARG 97 11.98 12.50 ARG99 12.16 12.50 ARG 101 14.07 12.50 ARG 119 16.80 12.50 ARG 169 13.5512.50 ARG 193 12.78 12.50 ARG 196 12.44 12.50 ARG 199 12.34 12.50 ARG222 13.63 12.50 ARG 234 12.80 12.50 ARG 243 12.36 12.50 ARG 247 12.2712.50 ARG 255 10.00 12.50 ARG 265 13.14 12.50 ARG 266 11.19 12.50 ARG307 12.83 12.50 ARG 310 13.21 12.50 ARG 324 12.66 12.50 ARG 335 12.1612.50 ARG 346 13.10 12.50 ARG 359 10.29 12.50 ARG 364 11.85 12.50 ARG375 12.65 12.50 ARG 379 12.22 12.50 ARG 380 12.33 12.50 ARG 381 12.4412.50 ARG 394 12.45 12.50 ARG 406 12.99 12.50 ARG 425 11.45 12.50 ARG460 11.44 12.50 ARG 465 12.42 12.50 ARG 482 10.68 12.50 ARG 484 12.3112.50 ARG 503 13.11 12.50 ARG 518 13.62 12.50 ARG 526 12.48 12.50 ARG585 12.11 12.50 ARG 606 13.59 12.50 ARG 612 12.73 12.50 ARG 613 12.4612.50 ARG 625 12.48 12.50 ARG 641 12.85 12.50 ARG 685 13.07 12.50 ARG689 13.04 12.50 ARG 694 12.46 12.50 ARG 713 12.20 12.50 ARG 743 12.3012.50 N+ 1 7.38 8.00

TABLE 6 Candidate amino acids for substitution in KOD DNA polymeraseAmino Acid pKa pKa Residue (calc) (model) ASP 4 7.06 3.80 ASP 6 −1.723.80 ASP 11 3.94 3.80 ASP 31 1.79 3.80 ASP 44 3.94 3.80 ASP 45 2.58 3.80ASP 92 2.18 3.80 ASP 98 3.41 3.80 ASP 108 3.11 3.80 ASP 113 −0.42 3.80ASP 123 1.89 3.80 ASP 132 3.23 3.80 ASP 141 15.80 3.80 ASP 164 3.36 3.80ASP 177 3.87 3.80 ASP 182 3.31 3.80 ASP 202 −1.29 3.80 ASP 204 1.17 3.80ASP 212 −3.07 3.80 ASP 215 4.76 3.80 ASP 235 −0.47 3.80 ASP 246 4.013.80 ASP 259 4.82 3.80 ASP 315 3.17 3.80 ASP 343 3.50 3.80 ASP 373 2.823.80 ASP 404 3.03 3.80 ASP 421 3.55 3.80 ASP 432 3.68 3.80 ASP 444 3.153.80 ASP 455 3.74 3.80 ASP 472 2.98 3.80 ASP 480 3.62 3.80 ASP 540 3.093.80 ASP 542 8.78 3.80 ASP 552 3.53 3.80 ASP 598 3.43 3.80 ASP 614 −1.003.80 ASP 633 3.59 3.80 ASP 635 3.18 3.80 ASP 718 −2.60 3.80 ASP 721−4.20 3.80 ASP 728 −5.80 3.80 ASP 754 −7.40 3.80 GLU 10 4.03 4.50 GLU 223.47 4.50 GLU 25 4.06 4.50 GLU 29 3.91 4.50 GLU 35 3.43 4.50 GLU 49 3.794.50 GLU 50 4.50 4.50 GLU 57 4.71 4.50 GLU 69 3.54 4.50 GLU 81 3.98 4.50GLU 102 4.78 4.50 GLU 111 2.22 4.50 GLU 130 4.78 4.50 GLU 133 4.36 4.50GLU 134 4.50 4.50 GLU 143 9.42 4.50 GLU 148 4.64 4.50 GLU 150 5.76 4.50GLU 151 15.70 4.50 GLU 154 4.10 4.50 GLU 165 3.26 4.50 GLU 166 4.35 4.50GLU 187 0.05 4.50 GLU 189 4.21 4.50 GLU 200 4.43 4.50 GLU 224 4.64 4.50GLU 238 3.49 4.50 GLU 251 4.31 4.50 GLU 276 0.56 4.50 GLU 280 4.78 4.50GLU 288 4.61 4.50 GLU 293 4.50 4.50 GLU 294 3.98 4.50 GLU 300 4.85 4.50GLU 303 4.57 4.50 GLU 306 4.69 4.50 GLU 314 3.66 4.50 GLU 321 3.37 4.50GLU 325 4.50 4.50 GLU 330 6.95 4.50 GLU 354 3.49 4.50 GLU 363 1.46 4.50GLU 366 4.66 4.50 GLU 374 4.36 4.50 GLU 376 3.83 4.50 GLU 385 4.50 4.50GLU 391 4.64 4.50 GLU 393 3.84 4.50 GLU 398 3.91 4.50 GLU 426 2.90 4.50GLU 430 4.57 4.50 GLU 458 4.50 4.50 GLU 459 4.53 4.50 GLU 475 4.54 4.50GLU 508 3.90 4.50 GLU 511 4.19 4.50 GLU 519 4.57 4.50 GLU 527 4.04 4.50GLU 529 3.93 4.50 GLU 530 4.05 4.50 GLU 554 4.60 4.50 GLU 562 4.64 4.50GLU 576 4.47 4.50 GLU 578 5.53 4.50 GLU 580 4.12 4.50 GLU 599 4.05 4.50GLU 600 4.64 4.50 GLU 609 14.10 4.50 GLU 617 12.50 4.50 GLU 621 10.904.50 GLU 628 4.47 4.50 GLU 637 4.50 4.50 GLU 645 4.50 4.50 GLU 648 4.504.50 GLU 654 4.50 4.50 GLU 658 3.94 4.50 GLU 664 9.30 4.50 GLU 719 7.704.50 GLU 730 6.10 4.50 GLU 734 4.50 4.50 GLU 742 2.90 4.50 GLU 753 1.304.50 HIS 59 6.43 6.50 HIS 89 4.54 6.50 HIS 103 6.15 6.50 HIS 147 6.366.50 HIS 257 4.00 6.50 HIS 416 2.74 6.50 HIS 439 7.09 6.50 HIS 663 6.506.50 HIS 679 6.50 6.50 HIS 725 6.50 6.50 CYS 223 10.07 9.00 CYS 42899.99 9.00 CYS 442 99.99 9.00 CYS 506 6.50 9.00 CYS 509 16.78 9.00 TYR 710.67 10.00 TYR 30 10.00 10.00 TYR 37 17.98 10.00 TYR 39 12.92 10.00 TYR86 13.65 10.00 TYR 110 12.67 10.00 TYR 112 10.84 10.00 TYR 120 13.5310.00 TYR 146 10.06 10.00 TYR 162 10.41 10.00 TYR 180 10.00 10.00 TYR209 11.28 10.00 TYR 218 7.75 10.00 TYR 261 9.34 10.00 TYR 273 9.22 10.00TYR 279 13.15 10.00 TYR 291 10.00 10.00 TYR 311 16.84 10.00 TYR 32014.28 10.00 TYR 362 12.68 10.00 TYR 384 10.00 10.00 TYR 388 10.00 10.00TYR 402 17.93 10.00 TYR 409 12.25 10.00 TYR 431 9.81 10.00 TYR 481 11.7610.00 TYR 493 12.60 10.00 TYR 494 9.46 10.00 TYR 496 14.11 10.00 TYR 49715.66 10.00 TYR 499 9.84 10.00 TYR 505 8.20 10.00 TYR 520 11.19 10.00TYR 532 11.04 10.00 TYR 538 12.70 10.00 TYR 566 13.34 10.00 TYR 57910.65 10.00 TYR 583 13.57 10.00 TYR 594 10.60 10.00 TYR 653 9.87 10.00TYR 701 10.00 10.00 TYR 750 10.24 10.00 N+ 17.37 8.00 LYS 13 10.15 10.50LYS 20 10.22 10.50 LYS 21 9.87 10.50 LYS 27 10.36 10.50 LYS 43 10.4310.50 LYS 52 10.08 10.50 LYS 53 10.08 10.50 LYS 66 10.01 10.50 LYS 709.94 10.50 LYS 73 10.06 10.50 LYS 74 10.50 10.50 LYS 84 9.60 10.50 LYS99 10.50 10.50 LYS 118 11.22 10.50 LYS 124 9.94 10.50 LYS 174 10.4310.50 LYS 192 10.36 10.50 LYS 199 8.13 10.50 LYS 201 9.94 10.50 LYS 22010.29 10.50 LYS 221 10.22 10.50 LYS 225 10.50 10.50 LYS 240 10.01 10.50LYS 253 12.95 10.50 LYS 287 14.68 10.50 LYS 289 11.48 10.50 LYS 31710.23 10.50 LYS 324 10.08 10.50 LYS 360 11.55 10.50 LYS 371 9.51 10.50LYS 375 10.50 10.50 LYS 429 10.50 10.50 LYS 443 10.22 10.50 LYS 46210.50 10.50 LYS 466 10.22 10.50 LYS 468 10.29 10.50 LYS 477 10.50 10.50LYS 487 10.24 10.50 LYS 507 11.91 10.50 LYS 526 10.22 10.50 LYS 53110.15 10.50 LYS 535 10.36 10.50 LYS 557 10.01 10.50 LYS 565 10.50 10.50LYS 570 10.36 10.50 LYS 592 10.50 10.50 LYS 602 10.43 10.50 LYS 63210.36 10.50 LYS 638 10.15 10.50 LYS 726 9.87 10.50 ARG 17 15.49 12.50ARG 32 12.15 12.50 ARG 58 11.45 12.50 ARG 67 11.73 12.50 ARG 78 11.9412.50 ARG 97 12.43 12.50 ARG 101 12.08 12.50 ARG 119 17.00 12.50 ARG 16912.15 12.50 ARG 188 11.94 12.50 ARG 193 13.69 12.50 ARG 196 12.29 12.50ARG 222 14.49 12.50 ARG 234 14.17 12.50 ARG 243 12.50 12.50 ARG 24712.01 12.50 ARG 255 9.85 12.50 ARG 265 12.01 12.50 ARG 266 10.80 12.50ARG 307 12.15 12.50 ARG 310 12.22 12.50 ARG 335 8.74 12.50 ARG 346 11.1612.50 ARG 359 9.85 12.50 ARG 364 11.90 12.50 ARG 379 12.08 12.50 ARG 38011.96 12.50 ARG 381 12.15 12.50 ARG 394 12.50 12.50 ARG 406 12.39 12.50ARG 425 11.45 12.50 ARG 440 12.50 12.50 ARG 460 11.45 12.50 ARG 47612.22 12.50 ARG 482 11.15 12.50 ARG 484 12.22 12.50 ARG 501 12.22 12.50ARG 503 13.21 12.50 ARG 518 11.94 12.50 ARG 585 11.66 12.50 ARG 60612.29 12.50 ARG 612 12.50 12.50 ARG 613 12.50 12.50 ARG 625 12.29 12.50ARG 641 12.15 12.50 ARG 685 12.50 12.50 ARG 713 12.50 12.50 ARG 74312.50 12.50 ARG 746 11.94 12.50 ARG 751 12.50 12.50 ARG 756 12.01 12.50

TABLE 7 Candidate amino acids for modification in B103-type polymerasesTable 7 Amino Acid Substituted Amino Acid Substituted Residue Amino AcidResidue Amino Acid H58 R E290 A H73 R E293 A H74 R E311 A H103 R E319 AH146 R E322 A H153 R E331 A H336 R E335 A H370 R E338 A H458 R E343 AH482 R E352 A E11 A E28 A E359 A E43 A E371 A E50 A E405 A E72 A E416 AE81 A E417 A E148 A E463 A E154 A E466 A E158 A E483 A E159 A E505 AE161 A E512 A E168 A E517 A E216 A C7 S E236 A C19 S E238 A C103 S E241A C445 S E273 A C452 S E276 A C513 S E288 A C527 S

What is claimed is:
 1. A method for performing a sequencing reactioncomprising: incorporating a nucleotide thio-triphosphate at a terminusof an extension primer that is hybridized to a target nucleic acid,wherein a reaction mixture for incorporating the nucleotide includes atleast one deoxyribonucleotide thio-triphosphate and a polymerase enzyme;hydrolyzing a thio-pyrophosphate formed in the incorporation step with aphosphatase enzyme, wherein the at least one deoxyribonucleotidethio-triphosphate is resistant to hydrolysis by the phosphatase enzyme;sequestering a by-product of the hydrolysis of the thio-pyrophosphatewith a complexing agent; and identifying the nucleotide that isincorporated at the terminus of the extension primer.1
 2. The method ofclaim 1, wherein the phosphatase enzyme is a pyrophosphatase enzyme, andwherein the at least one nucleotide is resistant to hydrolysis by thepyrophosphatase enzyme.
 3. The method of claim 1, wherein thephosphatase enzyme is an alkaline phosphatase enzyme.
 4. The method ofclaim 1, wherein the at least one nucleotide comprises adeoxyribonucleotide-5′-γ[gamma]-thio-triphosphate.
 5. The method ofclaim 1, wherein the complexing agent binds an orthophosphate moiety. 6.The method of claim 5, wherein the orthophosphate is selected from amonobasic orthophosphate, a dibasic orthophosphate, a tribasicorthophosphate, a monobasic thiophosphate, a dibasic thiophosphate, anda tribasic thiophosphate.
 7. The method of claim 1, wherein a specificrate of incorporation of the nucleotide thio-triphosphate is at least95% of the specific rate of incorporation of an analogous nucleotidewithout the thio-phosphate moiety.
 8. The method of claim 1, wherein thepolymerase enzyme is a Bst polymerase.
 9. The method of claim 1, whereinthe polymerase enzyme is a variant of a Bst polymerase.
 10. The methodof claim 9, wherein the variant comprises an amino acid sequence atleast 80% identical to SEQ ID NO:
 22. 11. The method of claim 9, whereinthe variant comprises an amino acid sequence at least 80% identical toSEQ ID NO:
 23. 12. The method of claim 9, wherein the variant comprisesan amino acid sequence at least 80% identical to SEQ ID NO:
 24. 13. Themethod of claim 1, wherein the polymerase enzyme is a Thermococcus sp.9° N-7 DNA polymerase.
 14. The method of claim 1, wherein the polymeraseenzyme is a KOD polymerase.
 15. The method of claim 1, wherein thepolymerase enzyme is a B103 polymerase.
 16. The method of claim 1,wherein the polymerase has a reduced buffering capacity within a definedpH range relative to a corresponding unsubstituted polymerase.
 17. Themethod of claim 16, wherein the defined pH range is about pH 5.5 toabout pH 9.5.
 18. The method of claim 16, wherein the defined pH rangeis about pH 7 to about pH
 9. 19. The method of claim 1, furthercomprising: identifying the nucleotide that is incorporated at theterminus of the extension primer by detecting a hydrogen ion that isproduced upon incorporation of the nucleotide.
 20. The method of claim19, wherein detecting the hydrogen ion is performed using an array ofsensors having at least one reaction chamber operatively coupled to eachsensor in the array of sensors.
 21. The method of claim 20 wherein theincorporating the nucleotide is conducted in the at least one reactionchamber operatively coupled to each sensor in the array of sensors. 22.The method of claim 20, wherein the array of sensors is an array ofISFET sensors.
 23. The method of claim 20, wherein the array of sensorsis at least 10⁷ sensors.